Thermally Sensitive Protecting Groups for Cysteine, and Manufacture and Use Thereof

ABSTRACT

In a preferred embodiment, there is provided a protecting group for protecting the thiol side chain of a cysteine residue, the protecting group comprising a Diels-Alder cycloadduct of a furan and a maleimide, and optionally, a linker interposed between the thiol side chain and the Diels-Alder cycloadduct.

RELATED APPLICATIONS

This application is a continuation of prior U.S. application Ser. No.16/985,815 filed 5 Aug. 2020, which claims the benefit of 35 USC §119(e) to U.S. Provisional Application Ser. No. 62/883,332 filed 6 Aug.2019.

SCOPE OF THE INVENTION

The present invention relates to a protected cysteine residue having aprotecting group bonded to a thiol side chain thereof, and which isconfigured to permit deprotection at a preselected temperature. Thepresent invention also relates to a method for producing the protectedcysteine residue, and a method for synthesizing a peptide containing aplurality of cysteines and using the protected cysteine residues tosequentially generate a plurality of specific disulfide bonds.

BACKGROUND OF THE INVENTION

Peptide synthesis involves formation of amide bonds between multipleamino acids by the condensation reaction of the carboxyl group of oneamino acid with the amino group of another amino acid. To synthesize apeptide of specific amino acid sequence, solid phase peptide synthesismay be used to form a peptide chain using successive reaction andintroduction of preselected amino acids to a solid insoluble and poroussupport, which includes a polymeric resin bead having a reactive linkergroup for attaching a growing peptide chain. The reactive linker groupmay include an amino group, and the amino acids to be introduced to thesolid support may be protected on the N-terminus (and possibly the sidechain as needed), using known protecting groups, such astert-Butyloxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc)protecting group.

Solid phase peptide synthesis may involve repeated cycles of couplingand N-terminal deprotection reactions, with washing of the resin beadbetween each cycle. Specifically, a first N-terminus protected aminoacid is coupled to the amino group of the reactive linker group in thesolid support, and the amino acid is deprotected to leave the aminofunctional group of the amino acid available to form an amide bond witha second N-terminus protected amino acid. The cycles are repeated with apreselected sequence of amino acids, and until the peptide of thedesired length and sequence is formed. The peptide as formed is thencleaved from the solid support, isolated and purified, and may besubject to further treatment.

Aside from the primary peptide sequence which may be obtained with solidphase peptide synthesis and which will fold into defined secondarystructures, disulfide bonds play an important role in the organizationof proteins and peptides, such as determining the ternary structure.Formation of correct disulfide bonds between correct cysteine residuesmay thus facilitate formation of correct three-dimensional structure,especially in short peptides. Disulfide bonds may however presentchallenges in synthesizing peptides and proteins by way ofbiotransformations; while the linear sequence may be readily programmedinto DNA, the information regarding which disulfide bonds should beformed is often regulated by additional factors and is part of thepost-translational modifications.

It has been recognized that a synthesized peptide intended to functionas a natural peptide of the same amino acid sequence and with specificdisulfide bonds may require further treatment during and after peptidesynthesis to ensure that the synthesized peptide possesses the samenatural disulfide bonds. Previously, orthogonal protection of the thiolside chains (involved in forming disulfide bonds) of cysteine residueshave been used to effect selective deprotection and disulfide bondformation after peptide synthesis. Specifically, a crude peptide isformed with cysteine residues having their respective thiol side chainsprotected with a protecting group, such as acetamidomethyl, tert-butyl,3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl or trityl, and whichremains on the thiol side chains after peptide synthesis. By having eachpair of specific cysteine residues intended to form a disulfide bondhaving orthogonal protection independent from other pairs of cysteineresidues, it has been possible to successively deprotect different pairsof cysteine residues and introduce regioselectivity to disulfide bondformation.

Peptide synthesis may thus employ multiple pairs of cysteine protectinggroups, in cases where multiple cysteine bonds are desired. A bioactivepeptide ordinarily possesses both the linear amino acid sequence and thespecific disulfide bonds made in the naturally-occurring material, whichif present contribute to the three dimensional structure. For example,if there are 8 cysteines in a short peptide, these would make 4disulfide bonds, and there would be 70 (8!4) possible structurescontaining 4 disulfide bonds that can be generated, where only 1 is thedesirable compound. Such need for increasing degrees of orthogonalitybetween protecting groups increases the complexity of the peptide, andrequires specialist and fine-tuning of peptide synthesis conditions, aswell as iterative cycles of reaction and purification to isolate thedesired compound and remove the unwanted reagents. This is a slow,costly and inefficient process, which may lead to low yields and complexpurification problems, and which may effectively prohibit scalability toproduce larger amounts of the desired peptide.

An improvement may reside with use of a common class of protectinggroups that are removed under the same trigger and differ from oneanother only by the threshold level of that trigger. This may preferablyinvolve only a single reagent introduced to the reaction mixture, withslow increase of its concentration. An existing example involves use ofacid-sensitive end-caps, although such approach has two complications:the acidic conditions used for deprotection are not compatible with theconditions required to dimerize the cysteine residues, and the dynamicrange between the acid-sensitivity of the protecting groups isinsufficient to provide the required levels of selectivity, withchemistry relegated between a pH of 0 and 5, sufficient for no more thanthree different acid-sensitive protecting groups (e.g. triggered atpH=0, 2.5 and 5). However, 2.5 pH units may not provide sufficientdiscrimination in reaction rates to ensure complete selectivity, andbeing triggered at pH 5 may result in background cleavage at neutral pHand may also limit synthetic operations available to the chemist makingthe peptide.

It has thus been appreciated that known regioselective formation ofdisulfide bonds may be associated with greater costs, time andcomplexity and reduced yield and selectivity, often stemming from theneed to develop customized residues and protecting groups, and in theabsence of any general approach to providing orthogonal protection.

SUMMARY OF THE INVENTION

It is a non-limiting object of the present invention to provide aprotecting group for protecting a cysteine residue and permitting moreregioselective formation of multiple disulfide bonds during peptidesynthesis, and which includes a Diels-Alder cycloadduct of an optionallysubstituted furan and an optionally substituted maleimide, andoptionally a cyclization spacer for placement between the thiol sidechain and the Diels-Alder cycloadduct.

It is another non-limiting object of the present invention to provide aprotecting group for protecting a cysteine residue for use in peptidesynthesis, and which may permit deprotection in response to a singlephysical change, such as the temperature, without necessary requiringuse of multiple reagents.

It is another non-limiting object of the present invention to provide aprotecting group for protecting a cysteine residue for use in peptidesynthesis, and which may permit configuration to obtain a family ofdifferent protecting groups selected for deprotection at differenttemperatures, so as to allow the peptide synthesis in a single potwithout necessary requiring isolation and purification steps betweenformation of multiple disulfide bonds.

It is another non-limiting object of the present invention to provide aprotected cysteine residue for preparing a synthetic peptide or proteinhaving a three dimensional structure, and which may permit readyincorporation into synthetic peptide synthesis to form two or morepreselected disulfide bonds to facilitate achieving the threedimensional structure.

It is another non-limiting object of the present invention to provide aprocess for synthesizing a peptide or protein having two of morepreselected disulfide bonds, and which includes increasing a reactiontemperature to effect sequential deprotection of protected cysteineresidue pairs to form the preselected disulfide bonds, withoutnecessarily requiring use deprotection reagents or separation orpurification steps.

It is another non-limiting object of the present invention to provide aprocess for synthesizing a peptide having two of more preselecteddisulfide bonds, and which may permit formation of functioning peptideswith reduced production of by-products having disulfide bonds other thanthe preselected disulfide bonds, and which may be adopted for a largerscale production of a peptide of commercial value, such as insulin orthe conotoxins, or a library of disulfide-containing research peptides.

In one simplified aspect, the present invention provides a protectinggroup for protecting the thiol side chain of a cysteine residue, theprotecting group comprising a Diels-Alder cycloadduct, either the endoor exo diastereomer, or a mixture of the two, of a furan and amaleimide, and optionally, a linker interposed between the thiol sidechain and the Diels-Alder cycloadduct. It is to be appreciated that thefuran, the maleimide and the linker are optionally substituted.

In one aspect, the present invention provides a compound havingstructural formula 1 or 2:

wherein R is an electron withdrawing group or a leaving group; X and Yare independently of each other oxygen, sulfur, nitrogen or phosphorus;R₁ and R₂ are independently of each other hydrogen, hydroxyl, halo,alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, formyl, haloformyl, carbonyl, carboxyl,alkoxy, alkoxycarbonyl, (alkoxycarbonyl)oxy, carbamoyl, amino, amido,imino, imido, azo, cyanato, isocyanato, cyano, nitro, sulfanyl,thiocyanato or phosphono, each of which is optionally substituted; and nis an integer between 1 and 12, inclusive.

In another aspect, the present invention provides a protecting group forprotecting a cysteine residue, the protecting group having structuralformula 1 or 2:

wherein R is an electron withdrawing group or a leaving group; X and Yare independently of each other oxygen, sulfur, selenium, nitrogen orphosphorus; R₁ and R₂ are independently of each other hydrogen,hydroxyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aryl, aralkyl, heteroaryl, heteroaralkyl, formyl, haloformyl, carbonyl,carboxyl, alkoxy, dialkoxy, trialkoxy, alkoxycarbonyl,(alkoxycarbonyl)oxy, carbamoyl, amino, amido, ammonio, imino, imido,azido, azo, cyanato, isocyanato, nitroxy, cyano, isocyano, nitrosooxy,nitro, nitrosyl, (carbamoyl)oxy, sulfanyl, disulfanyl, alkylsulfanyl,sulfinyl, sulfonyl, sulfoamido, sulfino, thiocyanate, isothiocyanato,thioyl, methanethioyl, mercaptocarbonyl, hydroxyl(thiocarbonyl),dithiocarboxy, phosphanyl or phosphono; and n is an integer between 1and 12, inclusive. It is to be appreciated that R, R₁ and R₂ areoptionally substituted.

In yet another aspect, the present invention provides a protecting groupfor protecting a cysteine residue, the protecting group havingstructural formula 1 or 2:

wherein R is an electron withdrawing group or a leaving group; X and Yare independently of each other oxygen, sulfur, nitrogen or phosphorus;R₁ and R₂ are independently of each other hydrogen, hydroxyl, halo,alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, formyl, haloformyl, carbonyl, carboxyl,alkoxy, alkoxycarbonyl, (alkoxycarbonyl)oxy, carbamoyl, amino, amido,imino, imido, azo, cyanato, isocyanato, cyano, nitro, sulfanyl,thiocyanato or phosphono, each of which is optionally substituted; and nis an integer between 1 and 12, inclusive.

In one embodiment, R is an activated ester or acid; X is sulfur, oxygenor nitrogen; Y is oxygen or sulfur; R₁ and R₂ are independently of eachother alkyl, aryl, a halogen, an ether, a thioether, a dialkylamine ortrialkylammonium, an ester or an acid derivative thereof, or a ketone;and n is an integer between 1 and 9, inclusive.

In one embodiment, R₁ and R₂ are independently of each other hydrogen,hydroxyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aryl, aralkyl, heteroaryl, heteroaralkyl, formyl, haloformyl, carbonyl,carboxyl, alkoxy, alkoxycarbonyl, (alkoxycarbonyl)oxy, carbamoyl, amino,imino, imido, azo, cyanato, isocyanato, nitroxy, cyano, isocyano, nitro,sulfanyl, alkylsulfanyl, sulfinyl, sulfino, thiocyanate orisothiocyanato. In one embodiment, R₁ and R₂ are independently of eachother hydrogen, hydroxyl, halo, alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, formyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl,carbamoyl, amino, nitro or alkylsulfanyl.

In one embodiment, R₁ is hydrogen, hydroxyl, halo, alkyl, formyl,carbonyl, carboxyl, alkoxy, alkoxycarbonyl, amino or nitro. In oneembodiment, R₁ is hydrogen, nitro, halo or alkoxy, preferably, hydrogen,nitro, bromo, chloro, fluoro, nitro, methoxy or ethoxy, or morepreferably, hydrogen, nitro, bromo or methoxy. In one embodiment, R₂ isalkyl or aryl. In one embodiment. R₂ is methyl, ethyl, propyl, butyl orphenyl. In one embodiment, R₂ is p-methoxyphenyl, p-nitrophenyl orbenzyl.

In one embodiment, R₁ and R₂ are independently of each other hydrogen,hydroxyl, halo, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,formyl, carbonyl, carboxyl, alkoxy, amino or nitro, each of which isoptionally substituted. In one embodiment, R₁ is hydrogen, hydroxyl,halo, alkyl, formyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl, aminoor nitro, each of which is optionally substituted, and R₂ is hydrogen,alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of whichis optionally substituted. In one embodiment, R_(j) is hydrogen, nitro,halo or alkoxy, and R₂ is alkyl, aryl or heteroaryl, each of which isoptionally substituted. In one embodiment, R₁ is hydrogen, nitro, bromo,chloro, fluoro, methoxy or ethoxy, and R₂ is methyl, ethyl, propyl,butyl, phenyl, p-methoxyphenyl, p-nitrophenyl or benzyl.

In one embodiment, X and Y are independently of each other oxygen ornitrogen. In one embodiment, X is nitrogen and Y is oxygen.

In one embodiment, n is an integer between 1 and 4, inclusive.

In one embodiment, R being the electron withdrawing group or the leavinggroup is as defined below. In one embodiment, R is hydroxyl, alkyl,alkenyl or halo. In one embodiment, R is hydroxyl, chloro, methyl orallyl.

In one embodiment, R₁ is substituted aryl, preferably 4-methoxyphenyl,4-nitrophenyl, 4-fluorophenyl, 4-trifluoromethylphenyl, 4-cyanophenyl,4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 4-di-N,N-methylaminophenyl,4-(methoxycarbonyl)phenyl, 3-methoxyphenyl, 3-nitrophenyl,3-fluorophenyl, 3-trifluoromethylphenyl, 3-cyanophenyl, 3-chlorophenyl,3-bromophenyl, 3-iodophenyl, 3-di-N,N-methylaminophenyl or3-(methoxycarbonyl)phenyl. In one embodiment, R₂ is propargyl,alkylazido, 4-azidophenyl, 4-alkynylphenyl or 4-propargyphenyl. In oneembodiment, R is OH, Cl, 4-nitrophenoxyl, Br, succinyl or other leavinggroup.

It is to be appreciated that compound 1 or 2 preferably comprises aDiels-Alder cycloadduct portion which may exist in an endo or exostereoisomeric form. It is to be also appreciated that compound 1 or 2may include a mixture of the endo and exo stereoisomers in differentproportions. In one embodiment, compound 1 or 2 contains, or is purifiedto contain, a greater portion of the endo or exo stereoisomer,preferably the endo stereoisomer. In one embodiment, compound 1 or 2contains 90 weight % or more, preferably 95 weight % or more, morepreferably 97% or more, or most preferably 99% or more of the endo orexo stereoisomer, or preferably the endo stereoisomer, based on thetotal weight of compound 1 or 2.

In another aspect, the present invention provides a method for preparinga protecting group, preferably compound 1 or compound 2, the methodincluding conducting a Diels-Alder reaction between a furan ofstructural formula 5 and a maleimide of structural formula 6 to producea cycloadduct of structural formula 7:

wherein R₁, R₂ and Y are as defined herein in respect of compound 1 or2. It is to be appreciated that Y included in the furan of formula 5 orthe cycloadduct of formula 7 may additionally include one or morehydrogens or other substituents to satisfy the octet rule. Preferably,the furan of formula 5 is a hydroxymethyl furan, i.e., Y is oxygenbonded to a hydrogen (hydroxyl).

In one embodiment, the Diels-Alder reaction is conducted at a reactiontemperature between about 0° C. and about 160° C., preferably betweenabout 0° C. and about 120° C. or more preferably between about 0° C. andabout 90° C. in a solvent for a reaction time of between about 10seconds and about 96 hours, preferably between about 5 minutes and about72 hours or more preferably between about 15 minutes and about 48 hours.In one embodiment, the solvent is one or more of benzene, acetonitrile,chloroform, dichloromethane, tetrahydrofuran, DMSO, DMF, toluene,xylene, hydrocarbon solvents, dichloroethane, tetrachloroethane,dioxane, methanol and isopropanol.

It is to be appreciated that the cycloadduct of formula 7 may includeendo and exo cycloadducts. In one embodiment, the method furthercomprises separating the endo and exo cycloadducts, preferably usingthin layer chromatography, column chromatography, high performanceliquid chromatography (HPLC), cyclotron or crystallization from acrystallization solvent. In one embodiment, said separating the endo andexo cycloadducts comprises separating the endo and exo cycloadducts toobtain the endo cycloadduct.

In one embodiment, the method further comprises activating Y with anactivating reagent, preferably to obtain an activated compound orcompound 2. In one embodiment, the activated compound comprises anactivated ester or acid coupled to Y, or preferably, an acyl halide,carboxyl, succinamyl (2,5-dioxo-1-pyrrolidinyl) carbonic ester oralkoxycarbonyl coupled to Y, wherein said acyl halide comprises —F, —Cl,—Br or —I, or preferably, the acyl halide is chloroformyl orbromoformyl. In one embodiment, the activating reagent comprisesphosgene, diphosgene, triphosgene, 4-nitrophenylchlorofonnate orcarbonyl diimidazole.

In one embodiment, the method further comprises coupling a linker to theactivated compound or compound 2 to obtain compound 1, wherein thelinker is preferably a compound of structural formula 8:

wherein X, R and n are as defined above in respect of compound 1. In oneembodiment, one or both of X and R are alkylamino.

In an alternative embodiment, the linker comprises optionallysubstituted straight chain or branched alkyl having a pair offunctionalized ends, wherein one said end is functionalized with anucleophilic heteroatom and the other said end is functionalized withcarboxyl or protected or masked carboxyl, and wherein the alkyl of thelinker comprises 3 to 10 methylene groups between the functionalizedends. In one embodiment, the nucleophilic heteroatom is amino, hydroxylor thiol. In one embodiment, the linker is a cyclization spacer.

In yet another aspect, the present invention provides a cysteine residuefor use in peptide synthesis, preferably solid phase peptide synthesisor more preferably Fmoc or Boc solid phase peptide synthesis, thecysteine residue having structural formula 3:

wherein R₄ is Fmoc or Boc, and R₃ is a protecting group compatible withcoupling of the protecting group on the thiol side chain.

In one embodiment, R₃ is allyl, tert-butyldimethylsilyl or TBS,methoxymethyl or MOM, ethoxymethyl or EOM, methyl, p-methoxybenzyl orp-nitrobenzyl. In one embodiment. R₃ is not a thermally labile orhydrogenolysis-labile protecting group. In one embodiment, the cysteineresidue is selected for use with compound 1 or 2.

In one preferred embodiment, compound 3 having as R₄ Fmoc and as R₃allyl is prepared from a commercially available compound 4 throughsequential allyl protection and trityl deprotection;

It is to be appreciated that the cysteine residue may include anL-cysteine, a D-cysteine or a combination thereof.

In yet another aspect, the present invention provides a protectedcysteine residue for peptide synthesis, the protected cysteine residuehaving structural formula 9 or 10:

wherein R₁ to R₄, X, Y and n are as defined herein.

In yet another aspect, the present invention provides a protectedcysteine having structural formula 9 or 10:

wherein X and Y are independently of each other oxygen, sulfur, nitrogenor phosphorus; R₁ and R₂ are independently of each other hydrogen,hydroxyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aryl, aralkyl, heteroaryl, heteroaralkyl, formyl, haloformyl, carbonyl,carboxyl, alkoxy, alkoxycarbonyl, (alkoxycarbonyl)oxy, carbamoyl, amino,amido, imino, imido, azo, cyanato, isocyanato, cyano, nitro, sulfanyl,thiocyanato or phosphono, each of which is optionally substituted; R₃and R₄ are independently each other hydrogen or a protecting group; andn is an integer between 1 and 12, inclusive.

In one embodiment, R₃ and R₄ are independently each other hydrogen,alkyl, allyl, tert-Butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl(Fmoc), tert-butyldimethylsilyl (TBS), methoxymethyl (MOM), ethoxymethyl(EOM), p-methoxybenzyl or p-nitrobenzyl. In one embodiment, R₃ ishydrogen or allyl and R₄ is Fmoc.

In one embodiment, R is an electron withdrawing group selected tofacilitate coupling of the protecting group to the thiol side chain ofthe cysteine residue. In one embodiment, the electron withdrawing groupis selected to draw electrons from the adjacent carbonyl or the carbonatom thereof. In one embodiment, the electron withdrawing group isselected to draw electrons from the adjacent carbonyl to facilitate asubstitution reaction with the thiol side chain, wherein in thesubstitution reaction, the electron withdrawing group is replaced by thethiol side chain. In one embodiment, the electron withdrawing group is agroup selected to reduce electron density of the moiety to which it isattached (relative to the density of the moiety without thesubstituent). In one embodiment, the electron withdrawing group isnitro, haloalkyl, halo, formyl, haloformyl, alkanoyl, alkylsulfonyl,cyano, alkylsulfinyl, carboxyl, alkoxycarbonyl, sulfonamido, amido,CONR¹⁰R²⁰, wherein R¹⁰ and R²⁰ are independently of each other hydrogen,alkyl, aryl, arylalkyl, heterocycloalkyl or cycloalkyl.

In one embodiment, R is a leaving group selected to facilitate couplingof the protecting group to the thiol side chain of the cysteine residue.In one embodiment, the leaving group is a species or moiety selected todetach from the protecting group during a reaction, such as asubstitution reaction. In one embodiment, the leaving group isdinitrogen, triflate, halogen, hydroxyl, amino, alkoxy, acyloxy(preferably —OAc, —OC(O)CF₃), sulfonate (preferably mesyl or tosyl),acetamide (preferably —NHC(O)Me), carbamate (preferably N(Me)C(O)Ot-Bu),phosphonate (preferably —OP(O)(OEt)₂) or alcohol.

In one embodiment, X is sulfur, oxygen or nitrogen, and Y is oxygen orsulfur, preferably, X is nitrogen and Y is oxygen.

In one embodiment, the term “alkyl” refers to a straight-chained orbranched hydrocarbon group containing 1 to 12 carbon atoms. The alkylmay include lower alkyl, referring to a C1-C6 alkyl chain. Examples ofthe alkyl group include methyl, ethyl, n-propyl, isopropyl, tert-butyl,and n-pentyl. The Alkyl group may be optionally substituted with one ormore substituents.

In one embodiment, the term “alkenyl” refers to an unsaturatedhydrocarbon chain that may be a straight chain or branched chain,containing 2 to 12 carbon atoms and at least one carbon-carbon doublebond. The Alkenyl group may be optionally substituted with one or moresubstituents. In one embodiment, the term “alkynyl” refers to anunsaturated hydrocarbon chain that may be a straight chain or branchedchain, containing 2 to 12 carbon atoms and at least one carbon-carbontriple bond. The alkynyl groups may be optionally substituted with oneor more substituents. The sp² or sp carbons of the alkenyl or alkynylgroup may optionally be the point of attachment of the group.

In one embodiment, the term “alkylene” refers to an alkyl group that hastwo points of attachment, and may preferably include (C1-C6) alkylene.In one embodiment, the alkylene is methylene, ethylene, n-propylene orisopropylene.

In one embodiment, the term “amino” refers to a functional group havinga nitrogen atom bonded to two hydrogen atoms, where one or both of thehydrogen atoms may optionally be substituted, preferably but not limitedto, alkyl or aryl, i.e., the amino includes primary, secondary, tertiaryor quaternary amino. For instance, the amino includes alkylamino,dialkylamino or trialkylamino.

In one embodiment, the term “cycloalkyl” refers to a hydrocarbon 3-8membered monocyclic or 7-14 membered bicyclic ring system having atleast one non-aromatic ring. The Cycloalkyl group is optionallysubstituted with one or more substituents, and may be cyclopropyl,cyclopentyl, cyclohexyl, cyclobutyl, cycloheptyl, cyclooctyl, cyclononylor cyclodecyl.

In one embodiment, the term “heterocycloalkyl” refers to a nonaromatic5-8 membered monocyclic, 8-12 membered bicyclic or 11-14 memberedtricyclic ring system comprising 1-3 heteroatoms if monocyclic, 1-6heteroatoms if bicyclic or 1-9 heteroatoms if tricyclic, saidheteroatoms being O, N, S, B, P or Si. The heterocycloalkyl isoptionally substituted with one or more substituents. In one embodiment,the heterocycloalkyl is piperidinyl, piperazinyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, 4-piperidonyl, tetrahydropyranyl,tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl,thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone,1,3-dioxolane, tetrahydrofuranyl, tetrahydrothienyl or thiirene.

In one embodiment, the term “aryl” refers to a hydrocarbon monocyclic,bicyclic or tricyclic aromatic ring system, and which is optionallysubstituted with one or more substituents. In one embodiment, the arylis phenyl, naphthyl, anthracenyl, fluorenyl, indenyl or azulenyl.

In one embodiment, the term “aralkyl” refers to aryl attached to anothergroup by a (C1-C6)alkylene group. The aralkyl is optionally substituted,either on the aryl portion or the alkylene portion of the aralkyl, withone or more substituent. In one embodiment, the aralkyl is benzyl,2-phenyl-ethyl or naphth-3-yl-methyl.

In one embodiment, the term “heteroaryl” refers to an aromatic 5-8membered monocyclic, 8-12 membered bicyclic or 11-14 membered tricyclicring system having 1-4 ring heteroatoms if monocyclic, 1-6 heteroatomsif bicyclic or 1-9 heteroatoms if tricyclic, where the heteroatoms areindependently O, N or S, and the remainder ring atoms are carbon. Theheteroaryl is optionally substituted with one or more substituents.

In one embodiment, the heteroaryl is pyridyl, 1-oxo-pyridyl, furanyl,benzo[1,3]dioxolyl, benzo[1,4]clioxinyl, thienyl, pyrrolyl, oxazolyl,oxadiazolyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl,isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl,thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl,indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl,benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl,azaindolyl, imidazopyridyl, quinazolinyl, purinyl,pyrrolo[2,3]pyrimidinyl, pyrazolo[3,4]pyrimidinyl, and benzo[b]thienyl,3H-thiazolo[2,3-c][1,2,4]thiadiazolyl,imidazo[1,2-d]-1,2,4-thiadiazolyl, imidazo[2,1-b]-1,3,4-thiadiazolyl,1H,2H-furo[3,4-d]-1,2,3-thiadiazolyl,1H-pyrazolo[5,1-c]-1,2,4-triazolyl, pyrrolo[3,4-d]-1,2,3-triazolyl,cyclopentatriazolyl or pyrrolo[2,1b]oxazolyl.

In one embodiment, the term “heteroaralkyl” or “heteroarylalkyl” means aheteroaryl group attached to another group by a (C1-C6)alkylene. Theheteroaralkyl may be optionally substituted, either on the heteroarylportion or the alkylene portion of the heteroaralkyl, with one or moresubstituent. In one embodiment, the heteroaralkyl is2-(pyridin-4-yl)-propyl, 2-(thien-3-yl)-ethyl or imidazol-4-yl-methyl.

In one embodiment, the term “alkoxy” refers to an —O-alkyl radical.

In one embodiment, the term “ester” refers to a —C(O)OR³⁰, wherein R³⁰is preferably alkyl or aryl.

In one embodiment, the term “halogen” or “halo” is —F, —Cl, —Br or —I.In one embodiment, the term “haloalkyl” is an alkyl group in which oneor more hydrogen radicals are replaced by halogen, and may includeperhaloalkyl. In one embodiment, the haloalkyl is trifluoromethyl,difluoromethyl, bromomethyl, 1,2-dichloroethyl, 4-iodobutyl or2-fluoropentyl.

In one embodiment, the term “substituent” or “substituted” means that ahydrogen radical is replaced with a group that does not substantiallyadversely affect the stability or activity of the compound. The term“substituted” refers to one or more substituents, which may be the sameor different, each replacing a hydrogen atom. In one embodiment, thesubstituent is halogen, hydroxyl, amino, alkylamino, arylamino,dialkylamino, diarylamino, cyano, nitro, mercapto, oxo, carbonyl, thio,imino, formyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato,sulfoamido, sulfonylalkyl, sulfonylaryl, alkyl, alkenyl, alkoxy,mercaptoalkoxy, aryl, heteroaryl, cyclyl, heterocyclyl, wherein alkyl,alkenyl, alkyloxy, aryl, heteroaryl, cyclyl and heterocyclyl areoptionally substituted with alkyl, aryl, heteroaryl, halogen, hydroxyl,amino, mercapto, cyano, nitro, oxo, thioxo or imino.

It is to be appreciated that specific moieties recited in thedefinitions of the above variable groups, including, but not limited to,R and R₁ to R₄, may be optionally substituted.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference may now be had to the following detailed description takentogether with the accompanying drawings in which:

FIG. 1 is a reaction mechanism illustrating a process for synthesizing apeptide in accordance with a preferred embodiment of the presentinvention, and which involves solid-phase synthesis of the peptide witha protected cysteine residue and subsequent deprotection of theprotected cysteine residue to produce a free cysteine available to forma disulfide bond;

FIG. 2 is a scheme illustrating a process for synthesizing a peptide inaccordance with a preferred embodiment of the present invention, andwhich involves formation of two disulfide bonds at 60° C. and 100° C.;

FIG. 3 shows on the upper end, the example of a reverse Diels-Alderreaction for compound 188, and on the lower end a series of H NMRspectra taken of an initial sample of compound 188 after specifictimepoints (selected timepoints only). Specific protons in the startingmaterial and furna product are highlighted; the relative integration ofthe two indicated signals can be used to quantify the conversion of thereaction. This was cross-checked with three other signals and allmeasurements give the same values of conversion; and

FIG. 4 shows a deprotection curve showing release of a preferredprotecting group from a protected cysteine residue 188, with the Y-axisrepresents the percent release of the cysteine and the X-axis representsthe time in minutes. The Y values do not reach 100% due to the highconcentration of the experiment, and the restoration of the cyloadductfollowing release. Reaction is essentially complete after 40 minutes.This figure corresponds to the experimental series provided in FIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one aspect, the invention provides a set of cysteine building blocksfor solid-phase peptide synthesis designed for the selective formationof disulfide bonds after synthesis. They can be incorporated into normalpeptide synthesis using either Fmoc or Boc strategies (accountingfor >99% of all syntheses) without necessarily requiring any need formodification or customization.

It has been recognized disulfide bonds may be essential for biologicalactivity of peptides and proteins, and may assist organizing thethree-dimensional shape. The peptide synthesis gives us the linear orderof amino acids but does not in and of itself help us make thethree-dimensional shapes. To make the three-dimensional shapes, you needto make the disulfide bonds after the peptide synthesis. This mayrequire selectively deprotecting each pair of cysteines in turn, andthus requiring many different pairs of protecting group strategies ifyou have multiple disulfide bonds, as well as special reagents and veryexpensive, customized residues that need to be developed, tested, andevaluated for each specific case, as there are no general solutionscurrently.

This means everything must be redesigned every time, although it hasbeen proposed that escalating conditions can be used to controlselective deprotection. Acid-sensitive protecting groups are potentiallythe best candidates, with the idea being that a protecting group thatcleaves at pH 5 will cleave 100-times faster than one that cleaves at 3,which in turn will cleave 100-times faster than one that cleaves at 1.It has been recognized however that the problem with this approach isthat 100-times is often insufficient, and in this case you are limitedto three pairs of protecting groups; this means that this approach hasnot worked very well. Finally, there may be a lot of unintentionaldeprotection during synthesis because neutral conditions will stillresult in cleavage. The publication “Direct palladium-mediated on-resindisulfide formation from Allocam protected peptides”, Organic &Biomolecular Chemistry, 15.14 (2017): 2914-2918 to Stockdill reportsusing allylation chemistry to selectively deprotect cysteines, but therequired levels of discrimination were not possible to achieve, and thereaction requires a lot of some very expensive reagents (metallicpalladium) that need to be removed from the reaction mixture prior tobiological use.

Therefore, it has been appreciated that a preferred approach may allowfor a one-pot sequential and selective formation of the requireddisulfide bonds with no or less reagents needed, and no or lesspurifications or changes in the reaction mixture composition over thecourse of the reaction. It has been recognized that one possibility fora reagent-free approach may use heat. Most stable chemical bonds may notbe highly heat-sensitive, however, retro-cycloadditions can be highlytemperature sensitive, including Diels-Alder cycloadducts formed betweendienes and dienophiles.

The applicant has recognized that reactions between furans andmaleimides may be promising due to near-physiological temperatures whichmay be involved in the retro-Diels-Alder reaction. It has beenenvisioned that this may be due to a combination of a highly favoredforward reaction with a very low lying lowest unoccupied molecularorbital or LUMO for the maleimide, and a favorable reverse reaction asfuran is aromatic. It has also been envisioned that the precisethermodynamics and the temperature required to overcome the reversibleenergy barrier may be modulated through adjusting the electronics of thetransition states of the reaction, and by changing the substituents onthe furan and maleimide.

It has been envisioned that a process for synthesizing a peptide usingthe protected cysteine residue of the invention may be practiced“reagent-free” or with a reduced number of required reagents, and withuse of heat as the stimulus for deprotection and formation of disulfidebonds. To get around the background cleavage issue, it may be possibleto raise the initial temperature to 60° C., which may provide backgroundcleavage at ambient temperature (23° C.) at about 2000 times slower thanat 60° C. The following preferred non-limiting protected cysteineresidue may decompose at different temperatures to allow fordisassembly, while being reasonably stable at room temperature or atleast sufficiently stable to handle and use for peptide synthesis:

wherein R₁ is hydrogen, nitro, bromine or methoxy, and R₂ isp-methoxyphenyl, p-nitrophenyl or benzyl. The above preferrednon-limiting cysteine residue is protected with a protecting grouphaving two components, or namely a Diels-Alder cycloadduct of furan andmaleimide and a linker or cyclization spacer interposed between thethiol side chain and the Diels-Alder adduct.

It has been recognized that some background cleavage may remain if oneset of protecting groups cleave at 60° C., and the second at 80° C. So,the protecting group of the current invention most preferablyincorporates the Diels-Alder cycloadduct coupled with a second gatingmechanism, or namely a cyclization spacer. It has been recognized thatsmaller rings (for instance, down to 5 atoms) form faster than largerrings. So, a low temperature trigger coupled with a 5-membered spacermay liberate a cysteine much faster than a mid-temperature trigger witha 6-membered spacer (the 5-membered ring closes about 100-times fasterthan a 6-membered ring, which in turn is 100 times faster than a 7,which is about 100-times faster than an 8, which is 1000 times fasterthan a 9; differentiation between ring sizes may cease to significantlymatter much beyond this point). Consequently, even if the highertemperature thermally-active protecting group falls off at a lowertemperature than expected, the liberated cyclization spacer is lesslikely to cyclize, and the cysteine will not be easily liberated. Thisdual gating may permit each pair of cysteines to be liberated ordeprotected in turn, and will then dimerize to provide the disulfidebond before the next pair of cysteines is liberated.

The applicant has appreciated that consequently, the deprotectionreaction may proceed without the need for any change in conditions,except for a gradual increase in temperature. The products of thereactions may be innocuous, and may include for example a maleimide, afuran and a cyclic lactam, which may be readily separated from thepeptide by precipitation of the resulting peptide or an aqueous-organicextraction to remove the organic-soluble byproducts from the reactionmixture. A sacrificial amount of hindered thiol may be required toscavenge the maleimide if maleimide-thiol reactions are a possiblecomplication in specific cases, although the high dilution of thesereaction mixtures renders this an uncommon prospect as the two cysteinesin the molecule are held in close proximity.

It has been envisioned that while slow reaction rates of the differentcyclization spacers may potentially lead to problems, this may becounteracted by slowly increasing temperature, i.e., as the temperaturerises, the rates of reactions increase, and a spacer that is slow tocyclize at 60° C. will cyclize far faster at 80 or 100° C. Withdifferent combinations of the Diels-Alder cycloadduct with varyingsubstituents and the cyclization spacer with varying lengths, it may bepossible to devise a series of protecting groups that will be thermallytriggerable at 15 to 20° C. increments starting at 45° C., and allow forthe formation of 5 different systems, preferably triggerable at 45° C.,60° C., 75° C., 90° C. and 105° C., within reasonable temperatureranges. It is expected the higher temperatures may denature largeproteins, and the current invention may preferably encompass processesfor synthesizing peptides, such as insulin and the non-addictiveconotoxin pain-killers, where the higher temperatures are not expectedto be significantly problematic. The applicant has recognized that thehigher temperature ranges are often used during peptide synthesis in themicrowave reactors attached to many modern peptide synthesis machines.Finally, the cyclization could occur before cleavage from thesolid-support, further simplifying purification and improving yields.

Reference is made to FIG. 1 which illustrates a reaction pathway fordeprotection of the protected cysteine residue after peptide synthesis.First, a crude peptide was synthesized with Fmoc-protected amino acids,including an Fmoc-protected cysteine shown in FIG. 1 with a protectinggroup having a 7-oxanorbornene construct with R₁ and R₂, coupled with acyclization spacer having the variable length n. While not wishing to bebound by a theory, it has been appreciated that the 2-methylsubstitutedfuran ring forming part of the protecting group may possess naturalinstability which may be masked when incorporated as part of the7-oxanorbornene construct with maleimide, and with thermal decompositionof that construct, the unstable 2-methylsubstituted furan ring isremoved from the cyclization spacer.

Reference is made to FIG. 2 which illustrates a possible reactionpathway for formation of two disulfide bonds sequentially between a pairof protected thiol side chains “SR₁” and then between a pair of thiolside chains “SR₂”. For the sequential formation of the two disulfidebonds, a reaction mixture containing synthesized peptides incorporatingcysteine residues having pairs of SR₁ and SR₂ is heated first to about60° C. to effect deprotection of the pair of SR₁ and form a disulfidebond therebetween. Then the temperature is raised to 100° C. to do thesame with the two SR₂.

Reference is made to FIG. 3 which demonstrates that at XXX ° C. theendcap is essentially completely removed from the cysteine in YYYminutes and that this change can be readily monitored by nuclearmagnetic resonance.

Reference is made to FIG. 4 which demonstrates a typical experiment,like that in FIG. 3, showing the end-cap removal and release of freecysteine as a function of time. This particular example relates to thespectra provided in FIG. 3, compound 188 releasing the end-cap at YYY °C.

In another preferred embodiment, there is provided a thermally-sensitiveprotecting group provided with a cycloadduct of a S-substituted furfuralalcohol and an N-alkyl or N-aryl-substituted maleimide, and which may beconfigured to permit different thermal sensitive triggers from 40° C. to120° C., and which may permit attachment to a protected cysteine aminoacid for a solid-phase synthesis, such as Fmoc solid-phase synthesis.Again, heat may be an ideal trigger for selective deprotection anddisulfide bond formation, without necessarily requiring use of areagent, and which may be applicable to conditions with a greaterdynamic range, lower background cleavage under standard operations, andreduced interference with ideal reaction conditions for disulfide bondformation.

Preferably, the protecting group is compound having structural formula 1or 2 as shown below:

wherein R is an activated ester or acid or forms part thereof; X issulfur, oxygen or nitrogen; Y is oxygen or sulfur; R₁ and R₂ are alkyl,aryl, a halogen, an ether, a thioether, a dialkylamine,trialkylammonium, an ester or an acid derivative thereof, or a ketone;and n is an integer from 1 to 9. It is to be appreciated that compound 2is provided without a linker included with compound 1 for directattachment to a cysteine residue.

Compounds 1 and 2 were made through the Diels-Alder reaction between theappropriate hydroxymethyl furan and maleimide using temperatures ofreaction between 0 and 90° C., and solvents, such as benzene,acetonitrile, chloroform, dichloromethane, tetrahydrofuran, DMSO. DMF,toluene, xylene, hydrocarbon solvents, dichloroethane,tetrachloroethane, dioxane, methanol and/or isopropanol with a reactiontimes between 15 minutes and 48 hours. The endo and exo cycloadductswere separated using column chromatography, HPLC or crystallization froman appropriate solvent.

The group Y was then activated using a carbonate equivalent agent tomake a chloroformate or activated carbonate. Preferred reagents includephosgene, diphosgene, triphosgene, 4-nitrophenylchlorofornate, carbonyldiimidazole and others alike. The foregoing steps could provide for theactive protecting group precursor 2.

In one embodiment, the activated carbonate thus obtained was thentreated with an alkyl linker with both ends of the chainfunctionalized—one with a nucleophilic heteroatom (amine, hydroxyl orthiol) and the other with a carboxylic acid or protected carboxylicacid, or masked carboxylic acid. The number of methylene groups orsubstituted methylene groups between the two terminal functionalitiesmay be 3 to 10, inclusive.

Alternatively, the activated carbonate derivative of the chloroformatewas not further functionalized, in which case the activated carbonatewas used directly with the cysteine residue.

General Procedures and Materials

Solvents were purchased from Caledon Labs (Caledon, Ontario),Sigma-Aldrich (Oakville, Ontario) or VWR Canada (Mississauga, Ontario).Other chemicals were purchased from Sigma-Aldrich, AK Scientific,Oakwood Chemicals. Alfa Aesar or Acros Chemicals and were used withoutfurther purification unless otherwise noted.

Anhydrous toluene, tetrahydrofuran (THF), diethyl ether andN,N-dimethylformamide (DMF) were obtained from an Innovative Technology(Newburyport, USA) solvent purification system based on aluminium oxidecolumns. CH₂Cl₂, pyridine, acetonitrile, N,N-diisopropylethylamine(DIPEA) and NEt₃ were freshly distilled from CaH₂ prior to use. Purifiedwater was obtained from a Millipore deionization system. All heatedreactions were conducted using oil baths on IKA RET Basic stir platesequipped with a P1000 temperature probe. Thin layer chromatography wasperformed using EMD aluminum-backed silica 60 F254-coated plates andwere visualized using either UV-light (254 nm), KMnO₄, vanillin,Hanessian's stain, or Dragendorff's stain. Preparative TLC was doneusing glass-backed silica plates (Silicycle) of either 250, 500, 1000 or2000 μm thickness depending on application. Column chromatography wascarried out using standard flash technique with silica (Siliaflash-P60,230-400 mesh Silicycle) under compressed air pressure. Standard work-upprocedure for all reactions undergoing an aqueous wash involved backextraction of every aqueous phase, a drying of the combined organicphases with anhydrous magnesium sulphate, filtration either using vacuumand a sintered-glass frit or through a glass-wool plug using gravity,and concentration under reduced pressure on a rotary evaporator (Buchior Synthware). ¹H NMR spectra were obtained at 300 MHz or 500 MHz, and¹³C NMR spectra were obtained at 75 or 125 MHz on Bruker instruments.NMR chemical shifts (8) are reported in ppm and are calibrated againstresidual solvent signals of CHCl₃ (δ 7.26), DMSO-d₅ (δ 2.50), acetone-d₅(δ 2.05), or methanol-d₃ (δ 3.31). HRMS were conducted on a Waters XEVOG2-XS TOF instrument with an ASAP probe in CI mode. Peptide synthesiswas accomplished using a modified Focus XC-6RV from AAPPTEC controlledby a PC loaded with Focus-XC software. Lyophilization was accomplishedusing a Sharp Freeze −80° C./6 L lyophilizer from AAPPTEC equipped witha 10-sample manifold. HPLC purification was conducted using analyticalanalysis on either a Waters HPLC with a 2489 UV/Vis detector and 1525Binary HPLC pump; or a Varian ProStar UIPLC equipped with two 218 pumps,a 320 UV detector, 330 photodiode array detector, 351 RI detector, and a410 autosampler. Preparative HPLC was conducted using an InterchimpuriFlash 5.25 multi HPLC with four individual solvent pumps.

Synthesis of Cycloadducts: General Procedure for the Synthesis ofMaleamic Acids 5a, 6a, 7a:

All compounds, or namely N-(4-methoxyphenyl)maleamic acid 5a,N-(4-nitrophenyl)maleamic acid 6a and N-benzylmaleamic acid 7a weresynthesized using methodologies described in Sortino, M. et al.,N-Phenyl and N-phenylalkyl-maleimides acting against Candida spp.:Time-to-kill, stability, interaction with maleamic acids. Bioorgan. Med.Chem. 2008, 16 (1), 560-568. DOI:http://dx.doi.org/10.1016/j.bmc.2007.08.030, the entire content of whichis incorporated herein by reference, with yields ranging from 90% to91%. Briefly, maleic anhydride (S1) (500 mg, 5.2 mmol) and equimolaramounts of the required amine were combined in CHCl₃ (6 mL) and stirredfor 45 minutes as a precipitate formed. This precipitate was thenfiltered and washed with cold (4° C.) water. The analytical data forthese maleamic acids have been previously published, and the data wasconsistent with the published spectra, as shown in Sortino noted aboveand in Sortino, M. et al., Antifungal, cytotoxic and SAR studies of aseries of N-alkyl, N-aryl and N-alkylphenyl-1,4-pyrrolediones andrelated compounds. Bioorgan. Med. Chem. 2011, 19 (9), 2823-2834. DOI:http://dx.doi.org/0.1016/j.bmc. 2011.03.038, the entire content of whichis incorporated herein by reference.

General Procedure for Synthesis of Maleimides 5, 6 and 7:

The maleamic acids (5a, 6a or 7a, 4.7 mmol) were dissolved in 5 mL ofacetic anhydride along with sodium acetate (100 mg, 1.2 mmol). Themixture was heated for 2-4 h at 100° C. (exact reaction time depended onthe substituent), until the reaction was determined to be complete byTLC. The solution was then cooled, diluted with water, then extractedrepeatedly with ethyl acetate. The combined organics were dried withmagnesium sulfate, and filtered and concentrated in the usual fashion.The solid residue was then redissolved in a minimum amount of THF, andprecipitated through the dropwise addition of ice-cold ether. The solidwas recovered, resuspended in additional minimal THF, and precipitatedby addition into cold water. A final filtration provided the desiredmaleimides in 73% to 80% yield. Spectroscopic data is consistent withprevious reports, as noted in Sortino, M. et al., Antifungal, cytotoxicand SAR studies of a series of N-alkyl, N-aryl andN-alkylphenyl-1,4-pyrrolediones and related compounds. Bioorgan. Med.Chem. 2011, 19 (9), 2823-2834. DOI: http://dx.doi.org/10.1016/j.bmc.2011.03.038.

N-benzyl-Maleimide 7

Prepared as per the general procedure above using 1 g of maleicanhydride (2-fold scale of the general protocol). Synthesis of 7aproceeded for 45 minutes, providing the maleimic acid in 93% crudeyield; the ring closing to 7 required only 1 hour. The crude mixture wasfirst purified by flash chromatography (7:3 hexanes-ethyl acetate), andthe fractions containing the product were combined, concentrated, andthen recrystallized from 2-propanol and water to provide an 80% yield ofthe title compound, as white crystals in 75% overall yield after vacuumdrying.

White crystals. ¹H NMR (300 MHz, CDCl₃) δ_(ppm): 7.33-7.22 (5H, m), 6.69(2H, s), 4.66 (2H, s). Spectral data are consistent with previouslypublished spectra in Sortino, M. et al., N-Phenyl andN-phenylalkyl-maleimides acting against Candida spp.: Time-to-kill,stability, interaction with maleamic acids. Bioorgan. Med. Chem. 2008,16 (1), 560-568. DOI: http://dx.doi.org/10.1016/j.bmc.2007.08.030.

N-(p-methoxyphenyl)-Maleimide 5

Prepared as per the general procedure above using 12.0 g of maleicanhydride (12-fold scale of the general protocol). Synthesis of 5aproceeded for 45 minutes, providing the maleimic acid in 90% crude yieldas a yellow powder. The ring closing (using 130 mL of acetic anhydrideand 5.8 g of sodium acetate) provided a dark yellow amorphous solid,that after recrystallization as in the general protocol, was recoveredas bright yellow needles in 73% yield: 66% yield overall from the maleicanhydride.

Yellow needles. ¹H NMR (300 MHz, CDCl₃) δ_(ppm): 7.27-7.23 (m, 2H),7.03-6.98 (m, 2H), 6.85 (s, 2H), 3.80 (s, 3H). Spectral data areconsistent with previously published spectra in Lee, H. S. et al.,Substituent chemical shifts of N-arylsuccinanilic acids,N-arylsuccinimides, N-arylmaleanilic acids, and N-arylmaleimides. Magn.Reson. Chem. 2009, 47 (9), 711-715. DOI: 10.1002/mrc.2450, the entirecontent of which is incorporated herein by reference.

N-(p-nitrophenyl)-Maleimide 6

Prepared as per the general procedure above using 10.0 g of maleicanhydride (10-fold scale of the general protocol). Synthesis of 6aproceeded for 2 hours, providing the maleimic acid in 91% crude yield asbrown crystals. The ring closing (using 115 mL of acetic anhydride and5.1 g of sodium acetate) provided a dark yellow amorphous solid, thatafter recrystallization as in the general protocol, was recovered as apale yellow powder in 78% yield; 71% yield overall from the maleicanhydride.

Yellow powder, ¹H NMR (300 MHz, CDCl₃) δ_(ppm): 8.35-8.32 (m, 2H),7.70-7.60 (m, 2H,), 6.94 (s, 2H). Spectral data are consistent withpreviously published spectra in Lee, H. S. et al., Substituent chemicalshifts of N-arylsuccinanilic acids, N-arylsuccinimides, N-arylmaleanilicacids, and N-arylmaleimides. Magn. Reson. Chem. 2009, 47 (9), 711-715.DOI: 10.1002/mrc.2450.

5-Nitrofurfural Diacetate, S3

Prepared according to a modified version of the protocol noted in Jin.H. et al., Lead optimization and anti-plant pathogenic fungi activitiesof daphneolone analogues from Stellera chamaejasme L. Pestic. Biochem.Physiol. 2009, 93 (3), 133-137. DOI:https://doi.org/10.1016/j.pestbp.2009.01.002, the entire content ofwhich is incorporated herein by reference. A mixture of 8.6 mLconcentrated HNO₃ and 0.06 mL concentrated H₂SO₄ was slowly added into90 mL of acetic anhydride while stirring at a temperature of 0° C. Thiswas followed by the slow addition of 10.4 mL of furfural, S2, into theacid mixture with stirring and temperature remaining at 0° C. Themixture was left to stir at this same temperature for 1 hour. At thistime, 80 mL of water was added and the mixture was left to stir at roomtemperature for an additional 30 minutes, over which time a whiteprecipitate formed. A 10% NaOH solution (10 g of NaOH in 100 mL ofwater) was then added to the mixture until the pH rose to 2.5. Themixture was then heated in a water bath at 55° C. for 1 hour. Aftercooling, the precipitate was filtered and washed with water prior tobeing recrystallized from anhydrous ethanol and dried to provide 5.2 gof white crystals in a 75% yield. The material was then used withoutfurther purification. R_(f)=0.37 (6:4, hexanes-ethyl acetate).

5-Nitrofurfural, S4

Prepared according to a modified version of the protocol noted in Jin,H. et al., Lead optimization and anti-plant pathogenic fungi activitiesof daphneolone analogues from Stellera chamaejasme L. Pestic. Biochem.Physiol. 2009, 93 (3), 133-137. DOI:https://doi.org/10.1016/j.pestbp.2009.01.002. Previously prepared5-nitrofufural diacetate (S3, 5.2 g, 21.4 mmol) was added to 52 mL of50% H₂SO₄ and the resulting mixture was heated using a heat gun (Wagnermodel #283022 HT 775, 540° C.) for 2 minutes. After cooling, thehydrolysate was extracted via ethyl acetate and the organic layer waswashed with water, dried with magnesium sulfate and then filtered andconcentrated. A simple distillation provided, after cooling of thedistillate, 2.5 g of the title compounds, S4, as a yellow-brownish solidin 83% yield.

Yellow-brownish solid; R_(f)=0.43 (1:1, hexanes-ethyl acetate); ¹H NMR(300 MHz, CDCl₃) δ_(ppm): 9.85 (s, 1H), 7.43 (d, J=3.82 Hz, 1H), 7.36(d, J=3.87 Hz, 1H). These obtained values are in agreement withpreviously reported spectroscopic data noted in Natarajan, P. et al.,Silver(I)-Promoted ipso-Nitration of Carboxylic Acids by NitroniumTetrafluoroborate. J. Org. Chem. 2015, 80 (21), 10498-10504. DOI:10.1021/acs.joc.5b02133, the entire content of which is incorporatedherein by reference.

5-Nitro-2-furanmethanol, 2

Prepared according to a modified version of the protocol of noted inEmami, S. et al., 7-Piperazinylquinolones with methylene-bridgednitrofuran scaffold as new antibacterial agents. Med. Chem. Res. 2013,22 (12), 5940-5947. DOI: 10.1007/s00044-013-0581-9, the entire contentof which is incorporated herein by reference. 5-nitrofurfural (S4, 2.22g, 15.7 mmol) was dissolved in 47 mL of absolute methanol and thesolution was cooled to 0° C. Then NaBH₄ (0.65 g, 0.017 mol) was slowlyadded and the solution was stirred for another 30 minutes. Once thereaction was complete, the solvent was removed under reduced pressureand the residue was then dissolved in a minimum amount of water. Thissolution was extracted with diethyl ether (3×10 mL). The combinedorganic phases were then washed with water, dried with magnesiumsulfate, filtered and concentrated. This provided 0.78 g of the titlecompound as a yellow oil in a moderate 35% yield.

Yellow oil; R_(f)=0.38 (1:1, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃) δ_(ppm): 7.28 (1H, d, J=3.71), 6.55 (1H, d, J=3.58), 4.70 (2H,s), 2.68 (1H, bs). These obtained values are in agreement withpreviously reported spectroscopic data noted in Berry, J. M. et al.,5-Nitrofuran-2-ylmethyl group as a potential bioreductively activatedpro-drug system. J. Chem. Sc. Perkin. Trans. 1 1997. (8), 1147-1156.DOI: 10.1039/a607202j, the entire content of which is incorporatedherein by reference.

Methyl 5-bromo-2-furoate S6

Prepared according to the approach noted in Torii, S. et al., AnodicReaction of 2-Furoic Acids. II. Electrolysis of Methyl5-Acetyl-2-furoate and Its Homologous in Protic Solvents. Bull. Chem.Soc. Jpn. 1972, 45 (9), 2783-2787. DOI: 10.1246/bcsj.45.2783, the entirecontent of which is incorporated herein by reference. Bromine (6.07 g,0.038 mol) was carefully added (dropwise over a period of 15 minutes) toa solution of methyl furoate (S5, 3.2 g, 0.025 mol) stirred at 50° C.under an argon atmosphere in a flame-dried round-bottom flask. Theresulting dark orange/brownish solution was additionally stirred foranother 15 minutes at 50° C. The reaction mixture was then poured intocold water (10 mL) and extracted with ethyl acetate (2×50 mL). Thecombined extracts were washed with water (1×10 mL) and brine (1×10 mL)prior to being dried with magnesium sulfate and concentrated. The finalproduct was purified by flash chromatography (10:1, hexanes-ethylacetate) to obtain 4.5 g of S6 in 85% yield. The spectral data wasconsistent with literature reports as noted in Torii above. R_(f)=0.17(7:3, hexanes-ethyl acetate).

5-Bromo-2-Furanmethanol 3

Prepared according to a modified version of the approach noted in Bi, J.et al., Application of furyl-stabilized sulfur ylides to a concisesynthesis of 8a-epi-swainsonine. Chem. Commun. 2008, (1), 120-122. DOI:10.1039/b713447a, the entire content of which is incorporated herein byreference. A stirred solution of methyl 5-bromo-2-furoate (S6, 6.7 g,4.6 mmol) in anhydrous THF (Ill mL) was cooled to 0° C. LiAlH₄ (1.4 g,5.1 mmol) was carefully added to the reaction mixture, which was thenstirred for a period of 15 minutes. Then, the reaction mixture waswarmed to room temperature over a period of 45 minutes prior to astandard Fieser quench and filtration (see (a) Fieser, L. F. et al.,Reagents for Organic Synthesis. Wiley: New York, 1976; p 1140: (b)Amundsen, L H. et al., Reduction of Nitriles to Primary Amines withLithium Aluminum Hydridel. J. Am. Chem. Soc. 1951, 73 (1), 242-244. DOI:10.1021/ja01145a082, the entire contents of both of which areincorporated herein by reference); the THF was mostly removed via rotaryevaporation. The resulting crude was diluted with ethyl acetate (200 mL)and washed with water (50 mL) and brine (50 mL). It was then dried overmagnesium sulfate and concentrated and the final product, 3.9 g of acolorless oil, was obtained after flash chromatography (5:1,hexanes-ethyl acetate) in 65% yield. Spectral data is consistent withthe published data, as noted in Bi above. R_(f)=0.50 (8:2, hexanes-ethylacetate).

Methyl 5-methoxy-2-furoate S7

Prepared according to a modified version of the protocol of Torii, S. etal., Anodic Reaction of 2-Furoic Acids. II. Electrolysis of Methyl5-Acetyl-2-furoate and Its Homologous in Protic Solvents. Bull. Chem.Soc. Jpn. 1972, 45 (9), 2783-2787. DOI: 10.1246/bcsj.45.2783. Methyl5-bromo-2-furoate (S6, 2.2 g, 10.7 mmol) was added to a solution ofsodium (0.6 g) and sodium iodide (0.03 g) in 30 ml of absolute methanol.The solution was refluxed for 7 hours, then poured into cold water (100mL). The mixture was then extracted thrice with diethyl ether, and thecombined organics were dried over magnesium sulfate, filtered andconcentrated. Chromatography of the resulting residue (8:2,hexanes-ethyl acetate) provided 0.8 g of the title compound as an oilyproduct in 48% yield.

Clear oil; R_(f)=0.50 (8:2, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃) δ_(ppm): 7.13 (d, J=3.69 Hz, 1H), 5.33 (d, J=3.60 Hz, 1H), 3.96(s, 3H), 3.86 (s, 3H). Spectral data is in agreement with published datanoted in Schwartz, D. A. et al., Synthetic approaches to haplophytine.2. Synthesis of4-methylamino-1-(2-furanyl)-2-phenyl-2-(2-pivaloylamidophenyl)butan-1-one.Can. J. Chem. 1983, 61 (6), 1126-1131. DOI: 10.1139/v83-201, the entirecontent of which is incorporated herein by reference.

5-methoxy-2-furanmethanol 4

Prepared according to a modified version of the protocol noted in Manly,D. G. et al., Simple Furan Ethers. II: 2-Alkoxy- and 2-Aryloxy-furans.J. Org. Chem. 1956, 21 (5), 516-519. DOI: 10.1021/jo01111a008, theentire content of which is incorporated herein by reference. A solutionof methyl 5-methoxy-2-furoate (S7, 0.45 g) in 2 mL of dry ether wasslowly added to a fast-stirring solution of LiAlH₄ (0.14 g) in 4.5 mL ofdry ether. After a 1.5 hour reflux, 0.3 mL of water was carefully addedprior to 1.9 mL of sodium hydroxide. The reaction mixture was dilutedwith ether, and the phases separated. The aqueous layer was thenextracted several times with ether, and the combined ether extracts weredried and evaporated to provide 150 mg of the title compound as acolorless liquid in 40% yield. No further purification was required.Colourless oil; R_(f)=0.21 (3:7, hexanes-ethyl acetate); ¹H NMR (300MHz, CDCl₃) δ_(ppm): 6.09 (d, J=3.30 Hz, 1H), 5.03 (d, J=3.30 Hz, 1H),4.37 (s, 21H), 3.78 (s, 3H), 2.89 (bs, 1H). Spectral data is inagreement with published data noted in Schwartz, D. A. et al., Syntheticapproaches to haplophytine. 2. Synthesis of4-methylamino-1-(2-furanyl)-2-phenyl-2-(2-pivaloylamidophenyl)butan-1-one.Can. J. Chem. 1983, 61 (6), 1126-1131. DOI: 10.1139/v83-201.

rac-(3aR,4R,7S,7aS)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(15_(endo)) andrac-(3aS,4R,7S,7aR)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(15_(exo))

N-(p-methoxyphenyl)-Maleimide 5 (0.5 g, 2.67 mmol) and 2-(hydroxymethyl)furan 1 (0.31 g, 3.2 mmol) were dissolved in anhydrous acetonitrileunder a nitrogen atmosphere in a flame-dried flask equipped with amagnetic stirring-bar. The reaction was stirred at 35° C. for 18 hours.When TLC indicated there was no longer starting material present, thesolvent was removed, and the reaction was concentrated under reducedpressure for 1 hour. Endo and exo cycloadducts was separated by column(6:4 to 4:6, hexanes-ethyl acetate). We obtained two fractions, onecontained 690 mg of pure endo material (85% yield), while the second(<5%) contained 26 mg of a mixture of the exo and endo isomers. Becausethese compounds have an inherently unstable nature at ambienttemperatures, they are kept stored at −20° C. until required.

rac-(3aR,4R,7S,7aS)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(15_(endo))

R_(f)=0.30 (2:8, hexanes-ethyl acetate); ¹H NMR (300 MHz, CD₃CN):δ_(ppm) 7.17-7.12 (m, 4H), 6.71 (dd, J=5.0, 1.5 Hz, 1H), 6.62 (dd,J=5.8, 1.6 Hz, 1H), 5.45 (d, J=6.7 Hz, 1H), 4.32 (dd, J=12.94, 5.67 Hz,1H), 4.20 (dd, J=12.82, 6.37 Hz, 1H), 3.95 (s, 3H), 3.93-3.85 (m, 1H),3.66 (d, J=7.67 Hz, 1H), 3.31 (t, J=5.88 Hz, 1H, OH); ¹³C NMR (75 MHz,CD₃CN): δ_(ppm) 175.0, 174.7, 159.7, 135.7, 135.4, 128.0, 124.9, 114.0,92.7, 79.5, 60.5, 55.3, 48.1, 45.7; HRMS (CI): Calculated for [M]⁺(C₁₆H₁₅NO₅): 301.0950. Found: 301.0944.

rac-(3aS,4R,7S,7aR)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(15_(exo))

R_(f)=0.19 (1:9, hexanes-ethyl acetate); ¹H NMR (300 MHz, CDCl₃):δ_(ppm) 7.07-6.89 (m, 4H), 6.65 (d, J=5.71 Hz, 1H), 6.60-6.55 (m, 1H),5.36 (d, J=1.65 Hz, 1H), 4.17-4.12 (m, 2H), 3.82 (s, 3H), 3.13 (d,J=6.57 Hz, 1H), 3.09 (d, J=6.58 Hz, 1H), 2.84 (bs, 1H, OH).

rac-(3aR,4R,7S,7aS)-4-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(16_(endo)) andrac-(3aS,4R,7S,7aR)-4-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(16_(exo))

N-(p-nitrophenyl)-Maleimide 6 (0.5 g, 2.30 mmol) and 2-(hydroxymethyl)furan 1 (0.273 g, 2.80 mmol) were dissolved in anhydrous acetonitrileunder a nitrogen atmosphere in a flame-dried flask equipped with amagnetic stirring-bar. The reaction was stirred at 40° C. After 4.5hours, a new polar spot formed but starting material was still presentand the reaction was left to stir at the same temperature overnight.When TLC indicated there was no longer starting material present, thesolvent was removed, and the reaction was concentrated under reducedpressure. A column with (7:3 to 3:7, hexanes-ethyl acetate) was used toseparate the components. The pure endo product was separated (100 mg,13%), while a mixture of the endo and exo products (300 mg, 39% yield)was obtained in a second fraction. Ultimately, there was a yield of 52%endo/exo cycloadduct mixture. Since this mixture has an inherentlyunstable nature at ambient temperatures, it is kept stored at −20° C.until required.

rac-(3aR,4R,7S,7aS)-4-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(16_(endo))

Light yellow solid; R_(f)=0.30 (3:7, hexanes-ethyl acetate): ¹H NMR (300MHz, CD₃CN): 8.29 (d, J=8.62 Hz, 2H), 7.42 (d, J=8.60 Hz, 2H), 6.60 (d,J=5.76 Hz, 1H), 6.49 (d, J=5.69 Hz, 1H), 5.34 (d, J=5.50 Hz, 1H), 4.18(dd, J=12.90, 5.80 Hz, 1H), 4.06 (dd, J=12.89, 6.30 Hz, 1H), 3.81 (dd,J=7.60, 5.61 Hz, 1H), 3.59 (d, J=7.75 Hz, 1H), 3.24 (t, J=6.08 Hz, 1H,OH); ¹³C NMR (75 MHz, CDCl₃): δ_(ppm) 174.1, 173.8, 147.4, 137.6, 135.8,135.6, 127.7, 124.4, 92.9, 79.6, 60.3, 48.3, 45.9; HRMS (CI): Calculatedfor [M]⁺ (C₁₅H₁₂N₂O₆): 316.0695. Found: [M]⁺ 316.0697.

rac-(3aS,4R,7S,7aR)-4-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(16_(exo))

White creamy solid; R_(f)=0.25 (3:7, hexanes-ethyl acetate); ¹H NMR (300MHz, CDCl₃): δ_(ppm) 8.32 (d, J=9.16 Hz, 2H), 7.58 (d, J=9.05 Hz, 2H),6.67-6.60 (m, 2H), 5.43 (d, J=1.22 Hz, 1H), 4.19 (d, J=7.29 Hz, 2H),3.24 (d, J=6.53 Hz, 1H), 3.19 (d, J=6.70 Hz, 1H), 2.54 (t, J=7.21 Hz,1H, OH)

rac-(3aR,4R,7S,7aS)-2-benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(17_(endo)) andrac-(3aS,4R,7S,7aR)-2-benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(17_(exo))

Made according to the protocol as noted in Fan, B. et al,Thermo-responsive self-immolative nanoassemblies: Direct and indirecttriggering. Chem. Commun. 2017, 0, 12068-12071. DOI:http://dx.doi.org/10.1039/c7cc06410a, the entire content of which isincorporated herein by reference. N-benzyl maleimide (7, 2.0 g, 10.7mmol) (see Sortino, M. et al., N-Phenyl and N-phenylalkyl-maleimidesacting against Candida spp.: Time-to-kill, stability, interaction withmaleamic acids. Bioorgan. Med. Chem. 2008, 16 (1), 560-568. DOI:http://dx.doi.org/10.1016/j.bmc.2007.08.030) and 2-(hydroxymethyl) furan(1, 931 μL, 1.05 g, 10.7 mmol) were dissolved in anhydrous acetonitrileunder a nitrogen atmosphere in a flame-dried flask equipped with amagnetic stirring-bar. The reaction was stirred at 35° C. for 14 hours.When TLC indicated the reaction had reached equilibrium, the solvent wasremoved, and the reaction was concentrated under reduced pressure for 1hour. Crude NMR indicated a ratio of (1:0.4:0.3) of endo-exo-unreactedmaleimide. The crude material was then purified by flash chromatography(6:4 to 4:6, hexanes-ethyl acetate) to provide 1.61 g (53% yield) of theendo and 677 mg (22% yield) of the exo product for a combined 75%isolated yield. Due to the inherent thermal instability, the material isstored at −20° C. until needed.

rac-(3aR,4R,7S,7aS)-2-benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(17_(endo))

Clear oil; R_(f)=0.27 (6:4, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃): δ_(ppm) 7.31-7.26 (m, 5H), 6.15 (dd, J=5.8, 1.5 Hz, 1H), 6.06(d, J=5.8 Hz, 1H), 5.26 (dd, J=5.5, 1.6 Hz, 1H), 4.47 (s, 2H), 4.25 (d,J=12.2 Hz, 1H), 4.15 (d, J=12.2 Hz, 1H), 3.63 (dd, J=7.6, 5.5 Hz, 1H),3.40 (d, J=7.6 Hz, 1H), 2.11 (s, 1H). Spectral data is consistent withthe published data noted in Fan, B. et al, Thermo-responsiveself-immolative nanoassemblies: Direct and indirect triggering. Chem.Commun. 2017, 0, 12068-12071. DOI: http://dx.doi.org/10.1039/c7cc06410a.

rac-(3aS,4R,7S,7aR)-2-benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(17_(exo))

Colourless solid; R_(f)=0.14 (6:4, hexanes-ethyl acetate); ¹H NMR (300MHz, CDCl₃): δ_(ppm) 7.33-7.26 (m, 5H), 6.61 (d, J=5.7 Hz, 1H), 6.54(dd, J=5.7, 1.5 Hz, 1H), 5.28 (d, J=1.7 Hz, 1H), 4.66 (bs, 2H), 4.09(dd, J=12.2, 8.8 Hz, 1H), 4.03 (dd, J=12.2, 6.3 Hz, 1H), 3.02 (d, J=6.5Hz, 1H), 2.99 (d, J=6.5 Hz, 1H), 2.76 (bt, J=7.4 Hz, 1H, OH). Spectraldata is consistent with the published data noted in Fan. B. et al,Thermo-responsive self-immolative nanoassemblies: Direct and indirecttriggering. Chem. Commun. 2017, 0, 12068-12071. DOI:http://dx.doi.org/10.1039/c7cc06410a.

rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-7-nitro-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(25_(endo)) andrac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-7-nitro-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(25_(exo))

N-(p-methoxyphenyl)-Maleimide 5 (0.349 g, 1.72 mmol) and 5-nitro-2furanmethanol 2 (0.295 g, 2.06 mmol) were dissolved in anhydrousacetonitrile under a nitrogen atmosphere in a flame-dried flask equippedwith a magnetic stirring-bar. The reaction was stirred at 50-75° C. for8 hours, at which time TLC analysis (3:7, hexanes-ethyl acetate)indicated the formation of a new, polar spot (R_(f)=0.13). The reactionwould not proceed to completion, and although we observed a new spotconsistent with the endo product form, it was negligible and was neverable to be isolated by chromatography. The reaction was stopped bycooling the mixture, and the solvent was removed. Column chromatography(6:4 to 2:8 to 0.5:9.5, hexanes-ethyl acetate) was carried out, andprovided 405 mg of the exo product in 63% yield. The material was keptat −20° C. until required.

rac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-7-nitro-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(25_(exo))

White spongy solid; R_(f)=0.13 (3:7, hexanes-ethyl acetate); ¹H NMR ¹HNMR (300 MHz, CDCl₃): δ_(ppm) 7.19 (d, J=9.03 Hz, 2H), 6.98 (d, J=8.98Hz, 2H), 6.96 (d, J=5.31 Hz, 1H), 6.90 (d, J=5.58 Hz, 1H), 4.22 (d,J=2.65 Hz, 1H), 4.19 (d, J=1.56 Hz, 1H), 3.83 (s, 3H), 3.77 (d, J=6.64Hz, 1H), 3.42 (d, J=6.64 Hz, 1H), 2.71 (t, J=7.46 Hz, 1H, OH); ¹³C NMR(300 MHz, MeOD): δ_(ppm) 172.8, 171.6, 160.2, 141.5, 134.8, 128.2,124.7, 114.4, 112.3, 91.8, 59.2, 55.1, 52.6, 50.9. HRMS (CI): Calculatedfor [M]⁺ (C₁₆H₁₄N₂O₇): 346.0801, [M+H]⁺ (C₆H₁₅N₂O₇): 347.0874. Found[M+H]⁺: 347.0877.

rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-nitro-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(26_(endo))

N-(p-nitrophenyl)-Maleimide 2 (0.254 g, 1.16 mmol) and5-nitro-2-furanmethanol 6 (0.200 g, 1.40 mmol) were dissolved inanhydrous acetonitrile under a nitrogen atmosphere in a flame-driedflask equipped with a magnetic stirring-bar. The reaction was stirred at40° C. for 4 hours, then at 60° C. for an additional 4 hours, at whichpoint only starting material was observed by TLC (3:7, hexanes-ethylacetate). The temperature was accordingly increased to 80° C., and wasleft to stir at this temperature overnight (12 hours). TLC indicatedmoderate conversion, and the solvent was removed, and the reactionmixture was concentrated. Column purification was then performed (6:4 to0.5:9.5, hexanes-ethyl acetate). 162 mg of the polar spot was obtainedin 31% yield (R_(f)=0.16) and was determined to be exo product. Anegligible amount of endo material was observed by crude NMR but was notable to be readily isolated from the starting materials. Product waskept stored at −20° C. until required.

rac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-7-nitro-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(26_(exo))

Off-white spongy solid; R_(f)=0.16 (3:7, hexanes-ethyl acetate); ¹H NMR(300 MHz, CD₃CN): δ_(ppm) 8.34 (d, J=9.05 Hz, 2H), 7.54 (d, J=9.11 Hz,2H), 6.93 (d, J=5.73 Hz, 1H), 6.90 (d, J=5.85 Hz, 1H), 4.26 (dd,J=13.21, 6.39 Hz, 1H), 4.05 (dd, J=13.32, 5.80 Hz, 1H), 3.94 (d, J=6.57Hz, 1H), 3.46 (d, J=6.70 Hz, 1H), 3.44 (t, J=5.93 Hz, 1H, OH); ¹³C NMR(100 MHz, CDCl₃): δ_(ppm) 171.7, 170.7, 147.6, 141.3, 137.0, 134.8,127.5, 124.6, 112.0, 91.7, 59.1, 52.7, 51.0. HRMS (CI): Calculated for[M]⁺ (C₁₅H₁₁N₃O₈): 361.0546; [M+H]⁺ (C₁₅H₁₂N₃O₈): 362.0624. Found [M]⁺:362.0614.

rac-(3aR,4R,7R,7aS)-2-benzyl-4-(hydroxymethyl)-7-nitro-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(27_(endo)) andrac-(3aS,4R,7R,7aR)-2-benzyl-4-hydroxymethyl)-7-nitro-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(27_(exo))

N-benzyl maleimide 7 (0.322 g, 1.72 mmol) and 5-nitro-2-furanmethanol 2(0.295 g, 2.06 mmol) were dissolved in anhydrous acetonitrile under anitrogen atmosphere in a flame-dried flask equipped with a magneticstirring-bar. The reaction was stirred at 65-70° C. for 16 hours. Atthat point. TLC analysis (2:8, hexanes-ethyl acetate) showed theformation a new nonpolar spot (endo; R_(f)=0.27) and the formation of apolar spot (exo: R_(f)=0.14). At this point, the reaction wasconcentrated under reduced pressure, and purified by flashchromatography (3:7 to 1:9, hexanes-ethyl acetate). The first fractioncontained 438 mg of product (69% yield) endo product and 129 mg (21%yield) of the exo product. They are both kept stored at −20° C. untilrequired.

rac-(3aR,4R,7R,7aS)-2-benzyl-4-(hydroxymethyl)-7-nitro-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(27_(endo))

White crystal; R_(f)=0.27 (2:8, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃): δ_(ppm) 7.35-7.27 (m, 5H), 6.46 (d, J=5.75 Hz, 1H), 6.23 (d,J=5.75 Hz, 1H), 4.55 (d, J=13.96 Hz, 1H), 4.50 (d, J=13.94 Hz, 1H), 4.29(dd, J=13.13, 6.18 Hz, 1H), 4.19 (dd, J=13.14, 6.94 Hz, 1H), 3.96 (d,J=7.95 Hz, 1H), 3.84 (d, J=7.95 Hz, 1H), 2.16 (t, J=6.60 Hz, 1H, OH);¹³C NMR (300 MHz, CDCl₃): δ_(ppm) 172.6, 170.7, 137.2, 134.9, 132.7,129.3, 128.8, 128.6, 112.4, 91.7, 60.8, 57.6, 51.0, 48.1, 43.0. HRMS(CI): Calculated for [M]⁺ (C₁₆H₁₄N₂O₆): 330.0852; [M+H]⁺ (C₁₆H₁₅N₂O₆):331.0930. Found [M+H]⁺: 331.0939.

rac-(3aS,4R,7R,7aR)-2-benzyl-4-hydroxymethyl)-7-nitro-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(27_(exo))

White crystal; R_(f)=0.14 (2:8, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃): δ_(ppm) 7.38-7.27 (m, 5H), 6.89 (d, J=5.62 Hz, 1H), 6.83 (d,J=5.52 Hz, 1H), 4.71 (d, J=14.23 Hz, 1), 4.63 (d, J=14.31 Hz, 1H),4.19-3.99 (m, 21H), 3.64 (d, J=6.48 Hz, 1H), 3.26 (d, J=6.47 Hz, 1H),2.75-2.68 (m, 1H, OH); ¹³C NMR (300 MHz, CDCl₃): δ_(ppm) 173.4, 170.6,141.3, 135.4, 134.9, 129.1, 128.7, 128.6, 111.4, 9.4, 60.3, 51.7, 50.9,43.5.

rac-(3aR,4S,7R,7aR)-4-bromo-7-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(35_(endo)) andrac-(3aS,4S,7R,7aS)-4-bromo-7-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1.3(2H)-dione(35_(exo))

N-(p-methoxyphenyl)-Maleimide 5 (0.8 g, 3.90 mmol) and5-Bromo-2-furanmethanol 3 (0.60 g, 3.00 mmol) were dissolved inanhydrous acetonitrile under a nitrogen atmosphere in a flame-driedflask equipped with a magnetic stirring-bar. The reaction was stirred atroom temperature for 3 hours, then the progress of reaction wasmonitored every 2 hours and the temperature was increased from 35° C. to75° C. over 6 hours, and held at the highest temperature for anadditional 10 hours. The reaction was concentrated under reducedpressure and purified by column (6:4 to 2:8, hexanes-ethyl acetate) toseparate the starting material from 851 mg of the non-polar endo(R_(f)=0.50, 6:4, hexanes-ethyl acetate) product (61% yield) and 63 mgof a polar exo (R_(f)=0.17, 6:4, hexanes-ethyl acetate) product in <5%yields. The mass balance included some debrominated material, 15 andstarting materials. These products kept stored at −20° C. untilrequired.

rac-(3aR,4S,71R,7aR)-4-bromo-7-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(35_(endo))

White solid. R_(f)=0.50 (6:4, hexanes-ethyl acetate); ¹H NMR (300 MHz,CD₃CN): δ_(ppm) 7.06-6.89 (m, 4H), 6.64 (d, J=5.54 Hz, 1H), 6.53 (d,J=5.44 Hz, 1H), 4.14 (dd, J=13.38, 6.13 Hz, 1H), 4.02 (dd, J=12.83, 6.25Hz, 1H), 3.87 (d, J=7.52 Hz, 1H), 3.79 (s, 3H), 3.74 (d, J=8.01 Hz, 1H),3.35 (t, J=5.99 Hz, 1H, OH); ¹³C NMR (75 MHz, CD₃CN): δ_(ppm) 174.9,174.0, 161.4, 140.9, 138.3, 129.7, 126.0, 115.9, 93.5, 89.8, 61.5, 58.2,56.8, 46.7. HRMS (CI): Calculated for [M]⁺ (C₁₆H₁₄BrNO₅): 379.0055.Found: 379.0046.

rac-(3aS,4S,7R,7aS)-4-bromo-7-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(35_(exo))

White solid; R_(f)=0.17 (6:4, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃): δ_(ppm) 7.20 (d, J=8.64 Hz, 1H), 6.97 (d, J=8.67 Hz, 1H), 6.64(d, J=7.74 Hz, 1H), 6.62 (d, J=7.89 Hz, 1H), 4.01 (d, J=11.56 Hz, 1H),3.84 (d, J=11.22 Hz, 1H), 3.83 (s, 3H), 3.36-3.33 (m, 2H); ¹³C NMR (75MHz, CDCl₃): δ_(ppm) 171.8, 170.5, 159.8, 142.1, 138.7, 127.6, 123.9,114.5, 89.4, 89.0, 55.5, 54.8, 51.5, 27.4.

rac-(3aR,4S,7R,7aR)-4-bromo-7-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(36_(endo))

N-(p-nitrophenyl)-Maleimide 6 (990 mg, 5.6 mmol) and5-Bromo-2-furanmethanol 3 (1.03 g, 4.7 mmol) were dissolved in anhydrousacetonitrile under a nitrogen atmosphere in a flame-dried flask equippedwith a magnetic stirring-bar. The reaction was stirred at 50° C. for 72hours until further conversion was no longer noted by TLC (6:4hexanes-ethyl acetate). The reaction mixture was concentrated, and theresidue purified by column chromatography (6:4, hexanes-ethyl acetate)in two columns successively (the first column provided productcontaminated with the starting materials). Crude 1H NMR had shown thepresence of both endo and exo derivatives, albeit in low conversion. Thesmall amount of exo was estimated to be well under 3% of the massbalance and was not isolated. The chromatography provided 530 mg of thetitle compound as a colourless solid in 26% yield.

rac-(3aR,4S,7R,7aR)-4-bromo-7-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(36_(endo))

White solid; R_(f)=0.20 (6:4, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃): δ_(ppm) 8.31 (d, J=8.75 Hz, 1H), 7.43 (d, J=8.48 Hz, 1H), 6.65(d, J=5.46 Hz, 1H), 6.51 (d, J=5.48 Hz, 1H), 4.36 (d, J=12.88 Hz, 1H),4.24 (d, J=13.02 Hz, 1H), 4.00 (d, J=7.90 Hz, 1H), 3.91 (d, J=8.00 Hz,1H); ¹³C NMR (75 MHz, CDCl₃): δ_(ppm) 171.9, 170.7, 147.4, 140.2, 136.0,127.1, 126.8, 124.6, 91.9, 87.6, 61.0, 56.8, 48.4. HRMS (CI): Calculatedfor [M]⁺ (C₁₅H₁₁BrN₂O₆): 393.9800, [M+H]⁺ (C₁₅H₁₂BrN₂O₆): 394.9879.Found: 394.9872.

rac-(3aR,4S,7R,7aR)-2-benzyl-4-bromo-7-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(37_(endo)) andrac-(3aS,4S,7R,7aS)-2-benzyl-4-bromo-7-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(37_(exo))

N-benzyl maleimide 7 (0.70 g, 3.8 mmol) and 5 Bromo-2-furanmethanol 3(0.80 g, 4.5 mmol) were dissolved in anhydrous acetonitrile under anitrogen atmosphere in a flame-dried flask equipped with a magneticstirring-bar. The reaction was stirred at room temperature for 7 hours,a TLC (7:3, hexanes-ethyl acetate) showing starting material was left,so the temperature was increased slowly (55′C to 70° C.) and theprogress of reaction was monitored every 2 hours. The reaction was leftfor an additional 14 hours at 70° C. when TLC showed some conversion.The solvent was removed, and the reaction mixture was concentrated.After column chromatography purification of reaction mixture (7:3,hexanes-ethyl acetate), 403 mg of an inseparable mixture of the endo andexo (2:1) cycloadducts as a white solid was obtained with an overallyield of 31%. In none of the more than 10 solvent mixtures examined wasseparation observed. The mixture was kept stored at −20° C. untilrequired.

rac-(3aR,4S,7R,7aR)-2-benzyl-4-bromo-7-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(37_(endo))

R_(f)=0.29 (7:3, hexanes-ethyl acetate); ¹H NMR (300 MHz, CDCl₃):δ_(ppm) 7.37-7.27 (m, 5H), 6.12 (d, J=5.51 Hz, 1H), 5.97 (d, J=5.24 Hz,1H), 4.52-4.46 (m, 2H), 4.24 (dd, J=12.40, 1.86 Hz, 1H), 4.13 (dd,J=12.44, 2.95 Hz, 1H), 3.79-3.12 (m, 2H), 3.25-3.20 (m, 1H, OH); ¹³C NMR(75 MHz, CDCl₃): δ_(ppm) 172.9, 172.2, 138.6, 135.2, 135.0, 129.1,128.6, 128.2, 90.6, 89.1, 68.9, 56.0, 51.6, 47.96, 42.5.

rac-(3aS,4S,7R,7aS)-2-benzyl-4-bromo-7-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(37_(exo))

R_(f)=0.28 (7:3, hexanes-ethyl acetate); ¹H NMR (300 MHz, CDCl₃):δ_(ppm) 7.37-7.27 (m, 5H), 6.12 (d, J=5.51 Hz, 1H), 5.97 (d, J=5.24 Hz,1H), 4.71 (d, J=14.35 Hz, 1H), 4.66 (d, J=14.37 Hz, 1H), 3.93 (d,J=11.41 Hz, 1H), 3.65 (dd, J=14.15, 7.79 Hz, 1H), 3.77-3.73 (m, 1H);3.25-3.20 (m, 1H, OR); ¹³C NMR (75 MHz, CDCl₃): δ_(ppm) 172.0, 170.9,139.2, 135.1, 135.0, 128.7, 128.3, 128.0, 90.5, 88.8, 69.1, 54.8, 48.04,42.9, 27.3.

Endo/exo mixture HRMS (CI): Calculated for [M]⁺ (C₁₆H₁₄BrNO₄): 363.0106,[M+H]⁺ (C₁₆H₁₅BrNO₄): 364.0184. Found: 364.0176.

rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(45_(endo)) andrac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(45_(exo))

N-(p-methoxyphenyl)-Maleimide 5 (0.32 g, 2.5 mmol) and5-methoxy-2-furanmethanol 4 (0.43 g, 2.1 mmol) were dissolved inanhydrous acetonitrile under a nitrogen atmosphere in a flame-driedflask equipped with a magnetic stirring-bar. The reaction was stirred at45° C. for 4 hours; TLC analysis (2:8, hexanes-ethyl acetate) indicatedthat a new nonpolar spot (R_(f)=0.50) had formed with some startingmaterial remaining. The temperature was then increased to 55-60° C. andthe reaction stirred for 14 hours. TLC showed a new polar spot(R_(f)=0.25) with no furan remaining. The solvent was then removed underreduced pressure. Column purification (4:6 to 1.5:8.5, hexanes-ethylacetate) was carried out to obtain 191 mg of the endo product (25%yield) and 26 mg (4% yield) of the exo product. Due to the fact thatthese compounds have an inherently unstable nature at ambienttemperatures, they are kept stored at −20° C. until required.

rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(45_(endo))

White solid; R_(f)=0.50 (2:8, hexanes-ethyl acetate); ¹H NMR (300 MHz,CD₃CN): δ_(ppm) 7.03 (d, J=9.10 Hz, 2H), 6.98 (d, J=9.14 Hz, 2H), 6.63(d, J=5.82 Hz, 1H), 6.58 (d, J=5.83 Hz, 1H), 4.10 (dd, J=12.91, 5.81 Hz,1H), 3.99 (dd, J=12.91, 6.13 Hz, 1H), 3.81 (s, 3H), 3.71 (d, J=7.87 Hz,1H), 3.56 (s, 3H), 3.51 (d, J=7.90 Hz, 1H), 3.20 (t, J=6.02, Hz, 1H,OH); ¹³C NMR (125 MHz, CDCl3): δ_(ppm) 174.5, 173.8, 159.8, 137.7,134.9, 128.2, 128.2, 124.9, 114.4, 86.8, 60.7, 55.3, 54.1, 50.2, 48.9.HRMS (CI): Calculated for [M]⁺ (C₁₇H₁₇NO₆): 331.1056, [M+H]⁺: 332.1134.Found [M+H]⁺: 332.1127.

rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(45_(exo))

White solid; R_(f)=0.25 (2:8, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃): δ_(ppm) 7.21 (d, J=8.94 Hz, 2H), 6.97 (d, J=9.01 Hz, 2H), 6.79(d, J=5.66 Hz, 1H), 6.56 (d, J=5.66 Hz, 1H), 4.14 (d, J=12.65 Hz, 1H),4.08 (d, J=12.71 Hz, 1H), 3.82 (s, 3H), 3.63 (s, 3H), 3.27 (d, J=6.59Hz, 1H), 3.21 (d, J:=6.57 Hz, 1H).

rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(46_(endo)) andrac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-7-methoxy-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(46_(exo))

N-(p-nitrophenyl)-Maleimide 6 (458 mg, 2.1 mmol) and5-methoxy-2-furanmethanol 4 (320 mg g, 2.5 mmol) were dissolved inanhydrous acetonitrile under a nitrogen atmosphere in a flame-driedflask equipped with a magnetic stirring-bar. The reaction was stirred at45° C. for 4 hours and then 80° C. for an additional 8; TLC analysis(2:8, hexanes-ethyl acetate) indicated that a new nonpolar spot(R_(f)=0.50) and polar spot had formed (R_(f)=0.21). The solvent wasthen removed under reduced pressure. Column purification (4:6 to1.5:8.5, hexanes-ethyl acetate) was carried out to obtain 400 mg of theendo product (52% yield) and approximately 30 mg of the exo product (4%yield). Due to the fact that these compounds have an inherently unstablenature at ambient temperatures, they are kept stored at −20° C. untilrequired.

rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(46_(endo))

White solid; R_(f)=0.50 (2:8, hexanes-ethyl acetate); ¹H NMR (300 MHz,CD₃CN): δ_(ppm) 8.30 (d, J=9.08 Hz, 2H), 7.43 (d, J=9.08 Hz, 2H), 6.65(d, J=5.87 Hz, 1H), 6.60 (d, J=5.81 Hz, 1H), 4.12 (dd, J=12.95, 5.84 Hz,1H), 4.01 (dd, J=12.90, 6.21 Hz, 1H), 3.80 (d, J=7.89 Hz, 1H), 3.59 (d,J=7.91 Hz, 1H), 3.57 (s, 3H), 3.24 (t, J=6.02 Hz, 1H, OH); ¹³C NMR (125MHz, CDCl₃): δ_(ppm) 173.5, 172.8, 147.5, 137.8, 137.6, 135.0, 127.7,124.4, 114.5, 86.9, 60.6, 54.2, 50.5, 49.1; HRMS (CI): Calculated for[M]⁺ (C₁₆H₁₄N₂O₇): 346.0801. [M+H]⁺ (C₁₆H₁₅N₂O₇): 347.0879. Found:347.0874.

rac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-7-methoxy-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(46_(exo))

R_(f)=0.21 (2:8, hexanes-ethyl acetate); ¹H NMR (300 MHz, CD₃CN):δ_(ppm) 8.48 (d, J=8.93 Hz, 2H), 7.69 (d, J=8.92 Hz, 2H), 6.89 (d,J=5.68 Hz, 1H), 6.79 (d, J=5.49 Hz, 1H), 4.29 (d, J=12.03 Hz, 1H), 4.11(d, J=12.77 Hz, 1H), 3.72 (s, 3H), 3.50-3.27 (m, 2H).

2-benzyl-4-(hydroxymethyl)-7-methoxyisoindoline-1,3-dione (47)

N-benzyl maleimide 7 (490 mg, 2.6 mmol) and 5-methoxy-2-furanmethanol 4(400 mg g, 3.0 mmol) were dissolved in anhydrous acetonitrile under anitrogen atmosphere in a flame-dried flask equipped with a magneticstirring-bar. The reaction was stirred at 40° C. for 4 hours, and 50° C.for 18 hours after which no reaction was observed. The reaction mixturewas then further heated to 65° C. for an additional 4 hours at whichpoint a new spot was observed by TLC (Rf=0.35, 2:8, hexanes-ethylacetate). The solvent was then removed under reduced pressure. Columnpurification (4:6 to 1.5:8.5, hexanes-ethyl acetate) was carried out toobtain 530 mg of a single product, the title compound, in 60% yield as awhite solid.

White solid. R_(f)=0.35 (2:8, hexanes-ethyl acetate); ¹H NMR (300 MHz,CDCl₃): δ_(ppm) 7.47 (d, J=8.58 Hz, 1H), 7.39-7.31 (m, 2H), 7.28-7.13(m, 3H), 7.05 (d, J=8.59 Hz, 1H), 4.78 (s, 2H), 4.74 (s, 2H), 3.93 (s,3H); ¹³C NMR (125 MHz, CDCl₃): δ_(ppm) 169.3, 166.5, 156.2, 136.2,135.9, 132.9, 130.9, 128.73, 128.65, 127.8, 117.6, 117.5, 61.9, 56.4,41.5. HRMS (CI): Calculated for [M]⁺ (C₁₇H₁₆NO₄): 297.1001, [M+H]⁺(C₁₇H₁₆NO₄): 298.1079. Found: 298.1075.

Synthesis of Protecting Groups: Synthesis of Benzyl-H Protecting GroupJT70B

(2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-yl)methyl-carbonochloridate(103)

2-Benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetra-hydro-1H-4,7-epoxyisoindole-1,3(2H)-dione(102) (0.25 g, 0.876 mmol) was dissolved in dry toluene (5.0 ml) undernitrogen. Once cooled to 0° C., N,N-diisopropylethylamine (0.46 ml) wasadded to the solution. Triphosgene (139.9 mg) was subsequently added tothe solution. The reaction was stirred under nitrogen for 2 hours at 0°C., and was then stirred for 12 hours at 25° C. The reaction mixture wasextracted once with ethyl acetate (10 ml). The organic fraction waswashed once with a solution of ammonium chloride (15 ml), and once morewith a solution of acidic brine (15 ml). The organic phase wassubsequently dried with anhydrous MgSO₄ and concentrated under reducedpressure to deliver the title compound (0.221 g, 75% yield). Rf 0.71 in3:7 hexane/ethyl acetate. ¹H NMR (300 MHz, CDCl₃) δ 7.42-7.24 (m, 10H),7.24-7.15 (m, 2H), 6.23 (dd, J=5.8, 1.7 Hz, 1H), 6.04 (d, J=5.8 Hz, 1H),5.42-5.26 (m, 1H), 5.13-4.97 (m, 1H), 4.91-4.63 (m, 2H), 4.52 (s, 2H),3.70 (dd, J=7.7, 5.5 Hz, 1H), 3.41 (d, J=7.7 Hz, 1H). ¹³C NMR (300 MHz,CDCl₃) δ_(ppm) 137.6, 137.2, 135.4, 135.1, 134.6, 129.1, 128.5, 125.3,90.4, 81.1, 79.6, 68.3, 62.7, 61.9, 50.2, 48.5, 47.9, 46.8, 42.4, 29.8,22.8, 21.7, 20.7, 16.0.

MS Calculated for C₁₇H₁₄ClNO₅ [M+H]⁺: 348.7500. Found (ASAP): 348.0639.

Methyl 4-aminobutanoate (104)

4-aminobutanoic acid (5.0 g, 48.5 mmol) was dissolved in methanol (75.0ml) and allowed to stir at 0° C. for 15 minutes. Thionyl chloride (5.27ml) was slowly added dropwise and the reaction was stirred at 0° C. for1.5 hours. The solvent was evaporated at reduced pressure and the titledcompound was delivered (4.97 g, 87%). ¹H NMR (300 MHz, CDCl₃) δ_(ppm)3.37 (s, 3H), 3.03 (t, J=7.3 Hz, 2H), 2.53 (t, J=7.9 Hz, 2H), 1.99 (p,J=7.5 Hz, 2H).

methyl4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-yl)methoxy)carbonyl)amino)butanoate(105)

(2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-yl)methyl-carbonchloridate(103) (500 mg, 1.63 mmol) was dissolved in chloroform (2.0 ml) undernitrogen, and cooled to 0° C. N,N-diisopropylethylamine (0.851 ml) wasthen added to the solution, followed by the addition of ethyl4-aminobutanoate (104) (228.8 mg, 1.956 mmol). The mixture was stirredunder nitrogen at room temperature for 6 hours. The reaction mixture wasdiluted with 10% hydrochloric acid (2 ml), and the resulting solutionwas extracted once with chloroform (2 ml). The organic fraction waswashed once more with saturated sodium bicarbonate (2 ml) and once morewith brine (2 ml). The resulting organic fraction was dried withanhydrous MgSO₄ and concentrated under reduced pressure in order toyield the titled compound as a waxy solid (302.1 mg, 43%) Rf 0.24 in 1:1hexane/ethyl acetate. ¹H NMR (300 MHz, DMSO) δ 7.39-7.12 (m, 5H), 6.59(d, J=5.7 Hz, 1H), 6.45 (d, J=5.7 Hz, 1H), 5.16 (d, J=1.8 Hz, 1H), 4.77(d, J=12.8 Hz, 1H), 4.57 (s, 2H), 4.15 (d, J=12.7 Hz, 1H), 3.57 (s, 2H),3.16 (d, J=6.4 Hz, 1H), 3.07 (s, 1H), 3.05-2.92 (m, 2H), 2.30 (t, J=7.4Hz, 2H). ¹³C NMR (75 MHz, DMSO) δ 174.53, 173.05, 155.75, 135.77,135.50, 134.54, 128.32, 127.81, 127.42, 89.78, 79.17, 78.78, 61.57,51.23, 47.43, 46.17, 41.40, 30.51, 28.35, 24.65, 10.79.

MS Calculated for C₂₂H₂₄N₂O₇ [M+H]⁺: 429.44. Found (ASAP): 429.1678.

4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-yl)methoxy)carbonyl)amino)butanoicacid (106)

Methyl4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-yl)methoxy)carbonyl)amino)butanoate(105) (200 mg, 0.467 mmol) was dissolved in 1,4-dioxane (0.92 ml) and 2NNaOH (4.00 ml). The resulting solution was stirred at 45′C for 24 hours,and subsequently cooled to room temperature. The reaction mixture wasdiluted with ethyl acetate (5 ml) and then acidified to pH 1 with thedropwise addition of 2N hydrochloric acid. Dichloromethane (5 ml) wasadded to the solution, and the organic and aqueous phases wereseparated. The aqueous fraction was stored at 0′C for 12 hours, and thetitled compound was obtained (100 mg, 53% yield) appearing as whitecrystals which formed in the aqueous layer. ¹H NMR (300 MHz, DMSO)δ_(ppm) 12.07 (s, 1H), 8.38 (s, 1H), 7.28 (m, 5H), 6.47 (d, J=4.9 Hz,1H), 6.26 (d, J=6.0 Hz, 1H), 5.09 (s, 1H), 4.58 (d, J=12.3 Hz, 1H), 4.32(m, 1H), 4.20 (m, 1H), 4.04 (d, 12.0 Hz, 1H), 2.98 (m, 2H), 2.81 (d,J=8.7 Hz, 1H), 2.64 (d, J=8.7 Hz, 2H), 1.60 (m, 2H).

Allyl 4-aminobutanoate (149)

At 0° C. and under nitrogen atmosphere, acetyl chloride (4.6 mL, 56.2mmol) was added dropwise into a flame dried flask of allyl alcohol (20mL). It was stirred it for at least 30 minutes then add 4-GABA (2.0 g,19.4 mmol) slowly. It was refluxed overnight then cooled and evaporatedin the morning under a ventilated fume hood. The concentrated mixturewas diluted with ethyl acetate then quenched with saturated sodiumbicarbonate. The organic layer was separated, and another extraction byethyl acetate was performed. The organic layer was then washed withbrine then dried with magnesium sulfate. A silica column was used topurify the crude at 4:6 methanol/ethyl acetate with the product beingthe first to elute. The titled compound ranged from light to dark brownoil (47%). Rf 0.7 in 4:6 hexane/ethyl acetate under vanillin stain. ¹HNMR (300 MHz, CDCl₃) δ 5.93-5.74 (m, 1H), 5.34-5.13 (m, 2H), 4.51 (dt,J=5.8, 1.4 Hz, 2H), 3.09 (t, J=7.6 Hz, 2H), 2.49 (t, J=7.2 Hz, 2H), 2.07(p, J=7.3 Hz, 2H).

Allyl4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-yl)methoxy)carbonyl)amino)butanoat(150)

The endcap (103) (722 mg, 2.08 mmol) was dissolved in 3 mL of chloroformand cooled to 0° C. DIPEA (1.7 ml) was subsequently added to thesolution and followed by addition of the allyl 4-aminobutanoate (357 mg,2.49 mmol). The mixture was stirred under nitrogen in room temperatureuntil starting material is consumed. The reaction mixture was washed thewith 10% HCl (3 ml) and was extracted with chloroform (3 ml) twice. Theorganic fraction was subsequently washed with saturated sodiumbicarbonate (4 ml) and once more with brine (4 ml). The resultingorganic fraction was dried with anhydrous MgSO₄ and concentrated underreduced pressure and the crude mixture was purified by silica columnchromatography in order to yield the title compound as a brown oil (39%)R_(f) 0.47 endo/0.29 exo in 1:1 hexane/ethyl acetate.

¹H NMR (300 MHz, CDCl3) δ 7.31-7.20 (m, 6H), 6.12 (dd, J=5.8, 1.6 Hz,1H), 6.02 (d, J=5.8 Hz, 1H), 5.88 (ddt, J=17.2, 10.4, 5.8 Hz, 1H), 5.22(ddt, J=11.7, 7.9, 1.4 Hz, 3H), 5.02 (s, 1H), 4.59-4.51 (m, 3H), 4.44(s, 2H), 3.59 (dd, J=7.7, 5.5 Hz, 1H), 3.21 (q, J=6.6 Hz, 2H), 2.36 (d,J=14.6 Hz, 2H), 1.82 (t, J=7.1 Hz, 2H). ¹³C NMR (76 MHz, CDCl₃) δ174.07, 173.86, 172.64, 155.63, 135.25, 135.17, 134.27, 131.97, 128.91,128.36, 127.91, 118.25, 89.97, 79.50, 65.09, 62.32, 47.52, 46.45, 42.21,40.34, 31.21, 30.78, 24.89.

MS Calculated for C₂₄H₂₆N₂O₇: 455.1818. Found (ASAP): 455.1817.

4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-yl)methoxy)carbonyl)amino)butanoicacid (151)

(Allyl4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-yl)methoxy)carbonyl)amino)butanoate(150) (50 mg, 0.11 mmol) was dissolved in THF (1.5 ml) in a flame driedround bottom flask under nitrogen atmosphere. Then morpholine (0.09 ml,1.1 mmol) and tetrakis(triphenylphosphine)-palladium (11.6 mg, 0.01mmol) were subsequently added. The reaction was left stirring at roomtemperature until starting material was consumed. The mixture wasfiltered and evaporated then washed with 10% HCl (1 ml) and extractedwith DCM. A column starting at 3:7 to 1:1 hexane/ethyl acetate to flushout the nonpolar components, then increased polarity to bring the titledcompound down with at least 7:3 hexane/ethyl acetate. The titledcompound was obtained (32% yield) appearing as yellow oil. Rf 0.19 in1:1 hexane/ethyl acetate. ¹H NMR (300 MHz, DMSO) δ_(ppm) 12.07 (s, 1H),8.38 (s, 1H), 7.28 (m, 51H), 6.47 (d, J=4.9 Hz, 1H), 6.26 (d, J=6.0 Hz,1H), 5.09 (s, 1H), 4.58 (d, J=12.3 Hz, 1H), 4.32 (m, 1H), 4.20 (m, 1H),4.04 (d, 12.0 Hz, 1H), 2.98 (m, 2H), 2.81 (d, J=8.7 Hz, 1H), 2.64 (d,J=8.7 Hz, 2H), 1.60 (m, 2H).

Synthesis of Protected Cysteines:

Allyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tritylthio)propanoate (200)

Fmoc-Cys(Trt)-OH (100 mg, 0.17 mmol) was dissolved in ethanol (1.25 ml)and an equimolar amount of cesium carbonate (55.4 mg, 0.17 mmol) wassubsequently added to the solution. The ethanol solvent was distilledoff at reduced pressure, and the remaining residue was taken up multipletimes by benzene and evaporated to dryness. The formed cesium salt wasdissolved in dimethylformamide (0.25 ml) and allyl bromide (0.34 ml, 4mmol) was subsequently added to the solution (225.2 mg, 1.86 mmol). Theresulting mixture was stirred at room temperature for 18 hours. Thesolvent was then evaporated under reduced pressure, and the crudemixture was purified by silica column chromatography in order to yieldthe title compound (76.6 mg, 72% yield) R_(f)0.44 in 1:4 hexane/ethylacetate.

¹H NMR (300 MHz, DMSO) δ 7.90 (p, J=10.0, 9.1 Hz, 3H), 7.72 (d, J=7.5Hz, 2H), 7.49-7.13 (m, 23H), 5.91-5.61 (m, 1H), 5.25-5.05 (m, 2H), 4.47(d, J=5.2 Hz, 2H), 4.36-4.09 (m, 3H), 3.86 (td, J=9.2, 4.8 Hz, 1H), 2.67(dd, J=12.8, 10.0 Hz, 1H). ¹³C NMR (75 MHz, DMSO) δ 170.29, 156.07,144.42, 144.03, 143.96, 141.00, 132.34, 129.37, 128.40, 127.92, 127.34,127.15, 125.51, 120.40, 117.71, 66.76, 66.04, 65.24, 53.79, 46.85,32.96. (Gaussian 3.40 Hz apodization)

Allyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-mercaptopropanoate(201)

Allyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tritylthio)propanoate(200) (100 mg, 0.16 mmol) was dissolved in DCM (2.0 ml) and stirred at0° C. for 15 minutes. Triethylsilane (23.0 mg, 0.19 mmol) was added tothe mixture, followed by the addition of TFA (0.2 ml). The mixture wasstirred at room temperature for 1 hour. The solvent was removed vianitrogen blowdown evaporation and the resultant crude mixture waspurified by silica column chromatography in order to yield the titlecompound (37.1 mg, 61% yield)

¹H NMR (300 MHz, CDCl3) δ 7.86-7.67 (m, 21H), 7.59 (s, 2H), 7.50-7.23(m, 4H), 5.99-5.66 (m, 1H), 5.42-5.17 (m, 1H), 4.68 (d, J=7.1 Hz, 2H),4.39 (d, J=7.2 Hz, 2H), 4.30-4.18 (m, 1H), 3.09 (dq, J=28.8, 17.2, 9.8Hz, 2H). ¹³C NMR (76 MHz, CDCl3) δ 170.89, 170.59, 156.42, 156.30,144.75, 144.28, 144.19, 144.05, 141.80, 134.49, 132.93, 131.81, 130.01,129.93, 128.78, 128.54, 128.25, 127.60, 127.42, 127.28, 126.79, 126.63,125.63, 125.34, 124.39, 120.50, 119.78, 119.66, 119.61, 119.28, 118.53,117.71, 67.88, 67.10, 67.00, 66.96, 66.63, 56.93, 55.73, 53.96, 53.31,47.65, 47.58, 47.54, 41.59, 35.17, 34.53, 33.15, 32.08, 31.21, 28.12,27.42, 25.79, 23.15, 14.62. (Gaussian 3.7 Hz appodization).

Coupling of Endcap Containing Various Linkers with Cysteines:

Fmoc-Cys-(S-Benzyl-maleimide)-(O-allyl) ((188)

Allyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-mercaptopropanoate(94.3 mg) was dissolved in THF (2 ml) and cooled in ice bath to 0° C. 3equimolar amount of DIPEA (0.06 ml) was subsequently added to thesolution, followed by the addition of endcap (103) (1 eq, 50 mg). Themixture was stirred under nitrogen at room temperature. The reactionmixture was diluted with 10% hydrochloric acid (10 ml), and theresulting solution was extracted with ethyl acetate (30 ml) two times.The organic fraction was washed once more with saturated sodiumbicarbonate (10 ml) and once more with brine (20 ml). The resultingorganic fraction was dried with anhydrous MgSO₄ and concentrated underreduced pressure and the crude mixture was purified by silica columnchromatography in order to yield the title compound as solid (76.6 mg,72% yield) R_(f)0.38 in 1:1 hexane/ethyl acetate. ¹H NMR (300 MHz,CDCl₃) δ 7.42-7.24 (m, 10H), 7.24-7.15 (m, 2H), 6.23 (dd, J=5.8, 1.7 Hz,1H), 6.04 (d, J=5.8 Hz, 1H), 5.42-5.26 (m, 1H), 5.13-4.97 (m, 1H),4.91-4.63 (m, 2H), 4.52 (s, 2H), 3.70 (dd, J=7.7, 5.5 Hz, 1H), 3.41 (d,J=7.7 Hz, 1H). ¹³C NMR (76 MHz, CDCl3) δ 174.35, 170.93, 156.32, 144.32,141.87, 136.65, 135.77, 134.78, 133.80, 131.91, 129.67, 129.47, 129.27,129.12, 128.97, 128.75, 128.29, 127.65, 126.59, 125.71, 120.55, 119.73,118.53, 89.51, 80.47, 68.75, 67.87, 67.04, 66.60, 60.96, 56.99, 54.00,48.06, 47.65, 46.94, 43.04, 33.29.31.31, 14.77.

Fmoc-Cys-(S-Benzyl-maleimide)-OH (216)

Fmoc-Cys-(S-Benzyl-maleimide)-(O-allyl) (188) (50 mg, 0.072 mmol) wasdissolved in THF (10 mL, 1 mmol) and morpholine (0.062 mL, 0.72 mmol).The resulting solution was stirred under nitrogen at room temperatureovernight, and subsequently added Pd(PPh₃)₄ (4.15 mg, 0.0036 mmol).After consumption of the starting material, the reaction mixture wasdiluted with 10% hydrochloric acid (5 ml), and the resulting solutionwas extracted with ethyl acetate (15 ml) two times and was washed oncemore with brine (10 ml). The resulting organic fraction was dried withanhydrous MgSO₄ and concentrated under reduced pressure and the crudemixture was purified by silica column chromatography in order to yieldthe title compound as solid (20 mg, 40% yield) Rf 0.2 in 1:1hexane/ethyl acetate.

¹H NMR (300 MHz, CDCl₃) δ 7.75-7.60 (m, 2H), 7.60-7.38 (m, 4H),7.38-7.22 (m, 13H), 6.62 (d, J=5.7 Hz, 1H), 6.54 (dd, J=5.7, 1.7 Hz,1H), 6.16 (dd, J=5.8, 1.6 Hz, 1H), 6.07 (d, J=5.8 Hz, 1H), 5.34-5.20 (m,2H), 4.67 (s, 2H), 4.48 (s, 2H), 4.34-3.98 (m, 4H), 3.64 (dd, J=7.7, 5.5Hz, 1H), 3.41 (d, J=7.6 Hz, 1H), 3.05-2.93 (m, 2H). ¹³C NMR (76 MHz,CDCl3) δ 175.83, 175.64, 174.89, 174.42, 138.44, 137.01, 135.40, 135.26,134.60, 132.19, 132.06, 131.99, 131.95, 129.08, 128.71, 128.61, 128.52,128.45, 128.14, 128.09, 127.94, 92.07, 91.46, 80.95, 79.63, 61.62,60.85, 50.02, 48.22, 48.04, 46.18, 42.63, 42.40, 29.72. (Gussain 1.60)

Fmoc-Cys-(S-4-aminobutyric-N-Benzyl-maleimide)-(O-allyl) (222)

Endcap (151) (67.7 mg, 0.216 ml) was dissolved in DMF (0.5 ml) andcooled to 0° C. 3 equimolar amount of DIPEA (0.06 ml) was subsequentlyadded to the solution, followed by the addition of EDC (38.53 mg, 0.273mmol) and HOBT (30.71 mg, 1.1 eq). The mixture was stirred undernitrogen for 10 minutes. ThenAllyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-mercaptopropanoate(201) (105.16 mg, 0.324 mmol) was added to the mixture and stirred undernitrogen overnight. The reaction mixture was diluted with ethyl acetate(20 ml) two times and then acidified with the 10% hydrochloric acid. Theorganic fraction was washed once more with saturated sodium bicarbonate(8 ml) and once more with brine (15 ml). The resulting organic fractionwas dried with anhydrous MgSO₄ and concentrated under reduced pressureand the crude mixture was purified by silica column chromatography inorder to yield the title compound as solid (11 mg, 16.2% yield) Rf 0.13in 1:1 hexane/ethyl acetate.

¹H NMR (300 MHz, CDCl₃) δ 7.76 (t, J=7.0 Hz, 3H), 7.69-7.50 (m, 3H),7.50-7.22 (m, 8H), 6.03-5.82 (m, 4H), 5.76 (d, J=8.2 Hz, 1H), 5.66 (d,J=8.3 Hz, 1H), 5.48-5.04 (m, 9H), 4.63 (dt, J=22.9, 5.9 Hz, 12H), 4.41(dq, J=12.8, 8.6, 6.4 Hz, 3H), 4.23 (dt, J=13.4, 7.1 Hz, 2H), 3.79 (d,J=4.4 Hz, 1H), 3.22-2.84 (m, 6H), 1.42 (p, J=5.7 Hz, 1H), 1.34-1.16 (m,2H).

Evaluation of Thermal Sensitivity of End-Caps: Details Regarding theKinetic Studies

In other experiments, kinetic studies were carried out in an NMR tube(see FIGS. 3 and 4). All samples were prepared in the same way. ForwardDiels-Alder reactions: 5.65 mg of furan (5.0 μL, 0.057 mmol) and anequimolar amount of the maleimide was added directly to 750 μL ofdeuterated solvent (either acetonitrile-d3 or more often DMSO-d6 for anyreaction 70° C. or above). For reverse Diels-Alder reactions, 16.0 mg ofcycloadduct was added to 3.0 mL of deuterated solvent in a vial. Thesolution was then partitioned between 4 NMR tubes and stored at −20° C.until used for an experiment. The experiments were carried out at theindicated temperatures on a Bruker 300 ¹H NMR spectrometer equipped witha variable temperature probe. A blank NMR tube containing the solventbut no analyte was inserted into the probe and the spectrometer wasallowed to equilibrate to the indicated temperature for 10 minutes. Thetube was then switched for the sample, and the experiment was run with aspectrum being collected every minute for the first 11 minutes, and thenevery 5 minutes thereafter for a total of four hours using themulti_zgvd script in the Bruker Topspin suite (8 scans per spectrum).Following data collection, the first and last spectra of the series wereexamined. If there was no change (and no appearance of starting materialor product as determined by comparison of the spectra with those ofpreviously isolated samples), no further action was taken, andconversion was determined to effectively be 0%. If any integration wasnoted in the final spectrum at the frequency corresponding to the peaksof the relevant reaction products, then all spectra in the series wereintegrated over identical frequency ranges. The data was exported to.txt files, imported into excel and converted to reaction conversions.Initial rates were calculated based on the first four data points. Inall observed cases, this region was effectively linear. The timerequired to obtain 25% conversion was determined by a simple linearinterpolation between the relevant data points. This value is arbitrary,but a half-life was considered to be a non accurate measurement as manyof the cycloreversions plateau before 50% conversion due to theequilibrium kinetics of the system. The 25% conversion point wasgenerally observed in the early stages of the cycloreversion and wasselected as a meaningful surrogate for our own use of these systems, andwill potentially play the same role for other researchers.

Synthesis of Linear Peptides:

The syntheses of the peptides were carried out using an AAPTTECFocusXC-6RV automated peptide synthesizer (6 reaction vessels), equippedwith an argon atmosphere, mechanical and gas-bubbling shaking systems,and a reaction vessel heating and/or cooling system controlled from anIBM PC using Focus XC software (v. 3.03). Preloaded Fmoc-Gly-Wang orFmoc-Ala-Wang resins were swollen in DMF for 1 hour followed byfiltration, and then were subjected to 20% piperidine in DMF twicesuccessively for 30 mins. Peptide couplings were carried out accordingto standard protocols for Fmoc solid phase synthesis using HCTU ascoupling agent (see Chan, W. C. et al., Basic procedures. In Fmoc SolidPhase Peptide Synthesis: A Practical Approach, Chan, W. C.; White. P.D., Eds. Oxford University Press: Oxford, 2000; pp 41-76, the entirecontents of which are incorporated herein by reference). All residueswere coupled using 5 equivalents of amino acid per functionalizedposition on the resin with 1 hour reaction times. All couplings werecarried out as double couplings. Following the coupling of each residuedeprotection of the Fmoc moiety was accomplished by treatment with 20%piperidine in DMF twice successively for 30 mins. Before and after eachcoupling, the beads were shaken 4 times with 4 mL of DMF followed byfiltration. Following the synthesis the beads were washed extensivelywith DMF (6×4 mL), MeOH (6×4 mL), DCM (6×4 mL), hexanes (6×4 mL), andfinally by ethanol (3×6 mL) and then removed from the synthesizer andstored in a desiccator under vacuum in the presence of P₂O₅ untilrequired. A small amount cleaved from the resin using 92.5:5:2.5 (v/v/v)TFA:triisopropylsilane:water. The peptide was purified using RP-HPLC(ramp from 0% to 18% acetonitrile in water over 5 mins followed byisocratic flow for 25 mins) unless otherwise stated.

While the invention has been described with reference to preferredembodiments, the invention is not or intended by the applicant to be solimited. A person skilled in the art would readily recognize andincorporate various modifications, additional elements and/or differentcombinations of the described components consistent with the scope ofthe invention as described herein.

We claim:
 1. A protected cysteine having structural formula 9 or 10:

wherein X and Y are independently of each other oxygen, sulfur, nitrogen or phosphorus; R₁ and R₂ are independently of each other hydrogen, hydroxyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, formyl, haloformyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl, (alkoxycarbonyl)oxy, carbamoyl, amino, amido, imino, imido, azo, cyanato, isocyanato, cyano, nitro, sulfanyl, thiocyanato or phosphono, each of which is optionally substituted; R₃ and R₄ are independently each other hydrogen or a protecting group; and n is an integer between 1 and 12, inclusive.
 2. The protected cysteine of claim 1, wherein R₁ and R₂ are independently of each other hydrogen, hydroxyl, halo, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, formyl, carbonyl, carboxyl, alkoxy, amino or nitro, each of which is optionally substituted.
 3. The protected cysteine of claim 1, wherein R₁ is hydrogen, hydroxyl, halo, alkyl, formyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl, amino or nitro, each of which is optionally substituted, and R₂ is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted.
 4. The protected cysteine of claim 1, wherein R₁ is hydrogen, nitro, halo or alkoxy, and R₂ is alkyl, aryl or heteroaryl, each of which is optionally substituted or wherein R₁ is hydrogen, nitro, bromo, chloro, fluoro, methoxy or ethoxy, and R₂ is methyl, ethyl, propyl, butyl, phenyl, p-methoxyphenyl, p-nitrophenyl or benzyl.
 5. The protected cysteine of claim 1, wherein X and Y are independently of each other oxygen or nitrogen or wherein X is nitrogen and Y is oxygen.
 6. The protected cysteine of claim 1, wherein n is an integer between 1 and 4, inclusive.
 7. The protected cysteine of claim 1, wherein R₃ and R₄ are independently each other hydrogen, alkyl, allyl, tert-Butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc), tert-butyldimethylsilyl (TBS), methoxymethyl (MOM), ethoxymethyl (EOM), p-methoxybenzyl or p-nitrobenzyl or wherein R₃ is hydrogen or allyl and R₄ is Fmoc. 